BACKGROUND
Technical Field
[0001] The present invention relates to a probe for detecting a polymorphism, a method of
detecting a polymorphism, a method of evaluating the efficacy of a drug, and a reagent
kit for detecting a polymorphism.
Related Art
[0002] The PIK3CA gene encodes the PI3 kinase which regulates a signaling pathway having
an important role in tumorigenesis. In tumor tissues, mutations in the PIK3CA gene
are observed in large numbers and amongst them, G1624A(E542K) mutation, G1633A(E545K)
mutation and A3140G(H1047R) mutation are observed at a high frequency (see, for example,
Science. (2004) Apr 23; 304(5670):554 (Non-patent Document 1) and
Oncogene. (2005) Feb 17; 24(8):1477-80 (Non-patent Document 2)).
[0003] It has been reported that, in cases where there is a mutation at the above-described
three positions, the therapeutic effects of an anti-EGFR antibody drug represented
by cetuximab, which is used as a therapeutic agent for unresectable colon cancer,
cannot be attained (see, for example,
Cancer Res. (2009); 69:(5). March 1, 2009 (Non-patent Document 3) and
BMC Cancer. (2011) Mar 25;11:107 (Non-patent Document 4)).
[0004] Molecular-targeted drugs such as cetuximab are very expensive and potentially cause
serious side-effects such as interstitial pneumonitis. Therefore, it is extremely
important to determine the gene mutation relevant to the efficacy of such a drug before
administration so as to selectively administer the drug to a patient expected to benefit
from the drug efficacy. In this manner, there have been demands for a method by which
a gene mutation is measured accurately in a short time as well as inexpensively and
easily.
[0005] At present, as a method of measuring a gene polymorphism, a method is known in which,
after amplifying a region containing a mutation by a PCR method, a nucleic acid probe
labeled with a fluorescent dye is hybridized to a target nucleic acid, and the decrease
in the amount of light emission by the fluorescent dye is measured so as to analyze
a mutation in a base sequence based on the results of melting curve analysis (see
Japanese Patent Application Laid-Open (JP-A) No.
2002-119291 (Patent Document 1)).
SUMMARY OF THE INVENTION
PROBLEMS TO BE SOLVED BY THE INVENTION
[0006] In Non-patent Documents 1 and 3, a mutation in PIK3CA is detected by a direct sequencing
method. However, such a direct sequencing method requires labour and cost because,
for example, after performing PCR, it is necessary to perform sequence reactions and
then electrophoresis using a sequencer. In addition, since it is required to process
the resulting amplification product, the amplification product may contaminate the
subsequent reaction systems.
[0007] In Non-patent Document 2, a mutation in PIK3CA is detected by using a PCR-SSCP (Single
Strand Conformation Polymorphism) method and a direct sequencing method in combination.
However, such a PCR-SSCP method requires labour and cost because, after performing
PCR, the resulting amplification product needs to be subjected to electrophoresis.
In addition, since it is required to process the amplification product, the amplification
product may contaminate the subsequent reaction systems.
[0008] In Non-patent Document 4, a mutation in PIK3CA is detected by the Scorpion ARMS method
using a kit manufactured by QIAGEN. However, for the Scorpion ARMS method, it is required
to purchase the kit. Further, in addition to the kit, a real-time PCR apparatus is
also required, increasing the cost. Moreover, since different reagents and reaction
tubes are necessary for each mutation, the labour and the cost are increased.
[0009] In Patent Document 1, a polymorphism is detected by a method utilizing a nucleic
acid probe. However, with regard to the design of the nucleic acid probe, the disclosure
offered in Patent Document 1 is merely that the nucleic acid probe is designed in
such a manner that, when a quenching probe whose terminus is labeled with a fluorescent
dye is hybridized to its target sequence, at least one G-C pair is formed by the multiple
base pairs of the hybrid which is generated between the nucleic acid probe and the
target sequence at the terminal. Therefore, in the method described in Patent Document
1, it is required to use a nucleic acid probe having an appropriate sequence for each
mutant type.
Furthermore, investigation by the present inventors revealed that the quenching probe
does not function sufficiently when the GC content is excessively high in the sequence
of the nucleic acid probe.
In view of these present circumstances, a further technical development for detecting
a polymorphism in the PIK3CA gene has been strongly desired.
[0010] An object of the present invention is to provide a polymorphism detection probe which
allows a polymorphism in the PIK3CA gene to be easily detected with high sensitivity
and a polymorphism detection method utilizing the probe. Another object of the present
invention is to provide a method of evaluating the efficacy of a drug in which the
above-described polymorphism detection method is used. Yet another object of the present
invention is to provide a reagent kit for detecting a polymorphism which includes
the above-described polymorphism detection probe.
MEANS FOR SOLVING THE PROBLEMS
[0011] The present inventors discovered that a polymorphism in the PIK3CA gene can be detected
by designing a quenching probe based on a specific region containing a polymorphism
in the PIK3CA gene and performing melting curve analysis using the quenching probe.
The present invention was achieved based on this discovery.
The present invention is as follows.
<1> A probe for detecting a polymorphism in the PIK3CA gene, which is at least one
fluorescently labeled oligonucleotide selected from the following P1 to P11':
(P1) an oligonucleotide having an identity of at least 80% to a base sequence having
a length of 7 to 50 bases including the 226th to the 232nd bases of the base sequence
indicated in any one of SEQ ID NOs: 1 to 3, wherein the base corresponding to the
226th base is a cytosine labeled with a fluorescent dye;
(P1') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence having a length of 7 to 50 bases including the 226th to
the 232nd bases of the base sequence indicated in any one of SEQ ID NOs: 1 to 3, wherein
the base corresponding to the 226th base is a cytosine labeled with a fluorescent
dye;
(P2) an oligonucleotide having an identity of at least 80% to a base sequence having
a length of 17 to 50 bases including the 139th to the 155th bases of the base sequence
indicated in SEQ ID NO:4 or 5, wherein the base corresponding to the 139th base is
a cytosine labeled with a fluorescent dye;
(P2') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence having a length of 17 to 50 bases including the 139th to
the 155th bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base
corresponding to the 139th base is a cytosine labeled with a fluorescent dye;
(P3) an oligonucleotide having an identity of at least 80% to a base sequence having
a length of 11 to 50 bases including the 155th to the 165th bases of the base sequence
indicated in SEQ ID NO:4 or 5, wherein the base corresponding to the 165th base is
a cytosine labeled with a fluorescent dye;
(P3') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence having a length of 11 to 50 bases including the 155th to
the 165th bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base
corresponding to the 165th base is a cytosine labeled with a fluorescent dye;
(P4) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 13 to 50 bases including the 155th to the 167th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 167th base is a cytosine labeled with a fluorescent dye;
(P4') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 13 to
50 bases including the 155th to the 167th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 167th base is a cytosine labeled
with a fluorescent dye;
(P5) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 10 to 50 bases including the 155th to the 164th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 164th base is a cytosine labeled with a fluorescent dye;
(P5') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 10 to
50 bases including the 155th to the 164th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 164th base is a cytosine labeled
with a fluorescent dye;
(P6) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 9 to 50 bases including the 155th to the 163rd
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 163rd base is a cytosine labeled with a fluorescent dye;
(P6') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 9 to
50 bases including the 155th to the 163rd bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 163rd base is a cytosine labeled
with a fluorescent dye;
(P7) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 7 to 50 bases including the 155th to the 161st
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 161st base is a cytosine labeled with a fluorescent dye;
(P7') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 7 to
50 bases including the 155th to the 161st bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 161st base is a cytosine labeled
with a fluorescent dye;
(P8) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 6 to 50 bases including the 155th to the 160th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 160th base is a cytosine labeled with a fluorescent dye;
(P8') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 6 to
50 bases including the 155th to the 160th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 160th base is a cytosine labeled
with a fluorescent dye;
(P9) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 12 to 50 bases including the 144th to the 155th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 144th base is a cytosine labeled with a fluorescent dye;
(P9') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 12 to
50 bases including the 144th to the 155th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 144th base is a cytosine labeled
with a fluorescent dye;
(P10) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 8 to 50 bases including the 148th to the 155th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 148th base is a cytosine labeled with a fluorescent dye;
(P10') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 8 to
50 bases including the 148th to the 155th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 148th base is a cytosine labeled
with a fluorescent dye;
(P11) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 5 to 50 bases including the 151 st to the 155th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 151 st base is a cytosine labeled with a fluorescent dye; or
(P11') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 5 to
50 bases including the 151 st to the 155th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 151st base is a cytosine labeled
with a fluorescent dye.
<2> The probe according to <1>, which is at least one fluorescently labeled oligonucleotide
selected from the following P1-1 to P11'-1:
(P1-1) an oligonucleotide having an identity of at least 80% to a base sequence having
a length of 7 to 50 bases including the 226th to the 232nd bases of the base sequence
indicated in any one of SEQ ID NOs: 1 to 3, wherein the base corresponding to the
226th base is a cytosine labeled with a fluorescent dye, the oligonucleotide recognizing
a polymorphism of at least one base selected from the group consisting of the 232nd
and the 241 st bases of SEQ ID NO:1;
(P1'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence having a length of 7 to 50 bases including the 226th to
the 232nd bases of the base sequence indicated in any one of SEQ ID NOs:1 to 3, wherein
the base corresponding to the 226th base is a cytosine labeled with a fluorescent
dye, the oligonucleotide recognizing a polymorphism of at least one base selected
from the group consisting of the 232nd and the 24 1 st bases of SEQ ID NO:1;
(P2-1) an oligonucleotide having an identity of at least 80% to a base sequence having
a length of 17 to 50 bases including the 139th to the 155th bases of the base sequence
indicated in SEQ ID NO:4 or 5, wherein the base corresponding to the 139th base is
a cytosine labeled with a fluorescent dye, the oligonucleotide recognizing a polymorphism
at the 155th base of SEQ ID NO:4;
(P2'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence having a length of 17 to 50 bases including the 139th to
the 155th bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base
corresponding to the 139th base is a cytosine labeled with a fluorescent dye, the
oligonucleotide recognizing a polymorphism at the 155th base of SEQ ID NO:4;
(P3-1) an oligonucleotide having an identity of at least 80% to a base sequence having
a length of 11 to 50 bases including the 155th to the 165th bases of the base sequence
indicated in SEQ ID NO:4 or 5, wherein the base corresponding to the 165th base is
a cytosine labeled with a fluorescent dye, the oligonucleotide recognizing a polymorphism
at the 155th base of SEQ ID NO:4;
(P3'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence having a length of 11 to 50 bases including the 155th to
the 165th bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base
corresponding to the 165th base is a cytosine labeled with a fluorescent dye, the
oligonucleotide recognizing a polymorphism at the 155th base of SEQ ID NO:4;
(P4-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 13 to 50 bases including the 155th to the 167th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 167th base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4;
(P4'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 13 to
50 bases including the 155th to the 167th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 167th base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4;
(P5-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 10 to 50 bases including the 155th to the 164th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 164th base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4;
(P5'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 10 to
50 bases including the 155th to the 164th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 164th base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4;
(P6-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 9 to 50 bases including the 155th to the 163rd
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 163rd base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4;
(P6'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 9 to
50 bases including the 155th to the 163rd bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 163rd base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4;
(P7-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 7 to 50 bases including the 155th to the 161st
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 161st base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4;
(P7'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 7 to
50 bases including the 155th to the 161st bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 161st base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4;
(P8-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 6 to 50 bases including the 155th to the 160th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 160th base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4;
(P8'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 6 to
50 bases including the 155th to the 160th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 160th base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4;
(P9-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 12 to 50 bases including the 144th to the 155th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 144th base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4;
(P9'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 12 to
50 bases including the 144th to the 155th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 144th base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4;
(P10-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 8 to 50 bases including the 148th to the 155th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 148th base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4;
(P10'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 8 to
50 bases including the 148th to the 155th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 148th base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4;
(P11-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 5 to 50 bases including the 151 st to the 155th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 151st base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4; or
(P11'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 5 to
50 bases including the 151 st to the 155th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 151st base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4.
<3> The probe according to <1> or <2>, wherein
the above-described P1 or P1' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 226th base labeled with a fluorescent dye at any one of the
first to the third positions from the 5'-end;
the above-described P2 or P2' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 139th base labeled with a fluorescent dye at any one of the
first to the third positions from the 5'-end;
the above-described P3 or P3' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 165th base labeled with a fluorescent dye at any one of the
first to the third positions from the 3'-end;
the above-described P4 or P4' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 167th base labeled with a fluorescent dye at any one of the
first to the third positions from the 5'-end;
the above-described P5 or P5' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 164th base labeled with a fluorescent dye at any one of the
first to the third positions from the 5'-end;
the above-described P6 or P6' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 163rd base labeled with a fluorescent dye at any one of the
first to the third positions from the 5'-end;
the above-described P7 or P7' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 161 st base labeled with a fluorescent dye at any one of the
first to the third positions from the 5'-end;
the above-described P8 or P8' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 160th base labeled with a fluorescent dye at any one of the
first to the third positions from the 5'-end;
the above-described P9 or P9' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 144th base labeled with a fluorescent dye at any one of the
first to the third positions from the 3'-end;
the above-described P10 or P10' fluorescently labeled oligonucleotide has the base
corresponding to the above-described 148th base labeled with a fluorescent dye at
any one of the first to the third positions from the 3'-end; or
the above-described P11 or P11' fluorescently labeled oligonucleotide has the base
corresponding to the above-described 151 st base labeled with a fluorescent dye at
any one of the first to the third positions from the 3'-end.
<4> The probe according to any one of <1> to <3>, wherein
the above-described P1 or P1' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 226th base labeled with a fluorescent dye at the 5'-end;
the above-described P2 or P2' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 139th base labeled with a fluorescent dye at the 5'-end;
the above-described P3 or P3' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 165th base labeled with a fluorescent dye at the 3'-end;
the above-described P4 or P4' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 167th base labeled with a fluorescent dye at the 5'-end;
the above-described P5 or P5' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 164th base labeled with a fluorescent dye at the 5'-end;
the above-described P6 or P6' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 163rd base labeled with a fluorescent dye at the 5'-end;
the above-described P7 or P7' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 161st base labeled with a fluorescent dye at the 5'-end;
the above-described P8 or P8' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 160th base labeled with a fluorescent dye at the 5'-end;
the above-described P9 or P9' fluorescently labeled oligonucleotide has the base corresponding
to the above-described 144th base labeled with a fluorescent dye at the 3'-end;
the above-described P10 or P10' fluorescently labeled oligonucleotide has the base
corresponding to the above-described 148th base labeled with a fluorescent dye at
the 3'-end; or
the above-described P11 or P11' fluorescently labeled oligonucleotide has the base
corresponding to the above-described 151 st base labeled with a fluorescent dye at
the 3'-end.
<5> The probe according to any one of <1> to <4>, wherein the above-described fluorescently
labeled oligonucleotides emit fluorescence when not hybridized to a target sequence,
and the fluorescence intensity of the fluorescently labeled oligonucleotides when
hybridized to the target sequence is decreased or increased as compared to when not
hybridized to the target sequence.
<6> The probe according to any one of <1> to <5>, which is a probe for melting curve
analysis. Alternatively expressed, use of a probe according to any one of <1> to <5>
for melting curve analysis.
<7> The probe according to any one of <1> to <6>, which has at least one base sequence
selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID
NO:21 and SEQ ID NO:22.
<8> A method of detecting a polymorphism in the PIK3CA gene, which includes the steps
of:
- (I) bringing the probe according to any one of <1> to <7> into contact with a single-stranded
nucleic acid contained in a sample to hybridize the above-described fluorescently
labeled oligonucleotide to the above-described single-stranded nucleic acid, thereby
obtaining a hybrid;
- (II) dissociating the above-described hybrid by changing the temperature of the sample
containing the hybrid to measure the change in fluorescence signal caused by dissociation
of the hybrid;
- (III) determining the Tm value, which is the dissociation temperature of the hybrid,
based on the above-described change in fluorescence signal; and
- (IV) based on the above-described Tm value, detecting the presence of a polymorphism
of the PIK3CA gene in the above-described single-stranded nucleic acid in the sample.
<9> The method according to <8>, which further includes the step of:
(V) based on the above-described presence of a polymorphism, determining the abundance
ratio of the single-stranded nucleic acid having the polymorphism in the single-stranded
nucleic acids contained in the above-described sample.
<10> The method according to <8> or <9>, which further includes the step of amplifying
the nucleic acid prior to or simultaneously with the above-described step (I) of obtaining
a hybrid.
<11> A method of evaluating the efficacy of an anticancer agent, which includes the
steps of:
detecting a polymorphism in the PIK3CA gene by the method of detecting a polymorphism
according to any one of <8> to <10>; and
determining (or predicting) the efficacy of the anticancer agent based on the detected
presence or absence of a polymorphism.
Said method may be performed on a sample from a subject (e.g. a human) in relation
to whom the evaluation is to be made. A sample (as used herein) comprises nucleic
acid (e.g. DNA) which may contain said polymorphism.
<12> A reagent kit for detecting a polymorphism in the PIK3CA gene, which includes
the probe according to any one of <1> to <7>.
<13> The reagent kit according to <12>, which further includes a primer for amplifying
a base sequence containing a region to which the above-described probe hybridizes.
The invention also extends to the use of the reagent kit according to <12> or <13>
or probe according to <1> to <7> for detecting said polymorphism in the PIK3CA gene.
EFFECTS OF THE INVENTION
[0012] According to the present invention, a polymorphism detection probe which allows a
polymorphism in the PIK3CA gene to be easily detected with high sensitivity and a
polymorphism detection method utilizing the probe are provided. In addition, the present
invention provides a method of evaluating the efficacy of a drug in which the above-described
polymorphism detection method is used. Further, the present invention also provides
a reagent kit for detecting a polymorphism which includes the above-described polymorphism
detection probe.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013]
Fig. 1A is an example of a melting curve of a nucleic acid mixture, and Fig. 1B is
an example of a differential melting curve of a nucleic acid mixture.
Fig. 2A to Fig 2N are differential melting curves of samples according to Example
1 of the present invention.
Fig. 3A to Fig 3L are differential melting curve of samples according to Example 2
of the present invention.
Fig. 4A to Fig. 4H are differential melting curves of samples according to Comparative
Example 1 of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The probe for detecting a polymorphism in the PIK3CA gene according to the present
invention (hereinafter, may be simply referred to as "the polymorphism detection probe")
is a probe for detecting a polymorphism in the PIK3CA gene which is at least one fluorescently
labeled oligonucleotide selected from the following P1 to P11':
(P1) an oligonucleotide having an identity of at least 80% to a base sequence having
a length of 7 to 50 bases including the 226th to the 232nd bases of the base sequence
indicated in any one of SEQ ID NOs: 1 to 3, wherein the base corresponding to the
226th base is a cytosine labeled with a fluorescent dye;
(P1') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence having a length of 7 to 50 bases including the 226th to
the 232nd bases of the base sequence indicated in any one of SEQ ID NOs: 1 to 3, wherein
the base corresponding to the 226th base is a cytosine labeled with a fluorescent
dye;
(P2) an oligonucleotide having an identity of at least 80% to a base sequence having
a length of 17 to 50 bases including the 139th to the 155th bases of the base sequence
indicated in SEQ ID NO:4 or 5, wherein the base corresponding to the 139th base is
a cytosine labeled with a fluorescent dye;
(P2') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence having a length of 17 to 50 bases including the 139th to
the 155th bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base
corresponding to the 139th base is a cytosine labeled with a fluorescent dye;
(P3) an oligonucleotide having an identity of at least 80% to a base sequence having
a length of 11 to 50 bases including the 155th to the 165th bases of the base sequence
indicated in SEQ ID NO:4 or 5, wherein the base corresponding to the 165th base is
a cytosine labeled with a fluorescent dye;
(P3') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence having a length of 11 to 50 bases including the 155th to
the 165th bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base
corresponding to the 165th base is a cytosine labeled with a fluorescent dye;
(P4) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 13 to 50 bases including the 155th to the 167th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 167th base is a cytosine labeled with a fluorescent dye;
(P4') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 13 to
50 bases including the 155th to the 167th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 167th base is a cytosine labeled
with a fluorescent dye;
(P5) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 10 to 50 bases including the 155th to the 164th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 164th base is a cytosine labeled with a fluorescent dye;
(P5') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 10 to
50 bases including the 155th to the 164th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 164th base is a cytosine labeled
with a fluorescent dye;
(P6) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 9 to 50 bases including the 155th to the 163rd
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 163rd base is a cytosine labeled with a fluorescent dye;
(P6') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 9 to
50 bases including the 155th to the 163rd bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 163rd base is a cytosine labeled
with a fluorescent dye;
(P7) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 7 to 50 bases including the 155th to the 161st
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 161st base is a cytosine labeled with a fluorescent dye;
(P7') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 7 to
50 bases including the 155th to the 161st bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 161st base is a cytosine labeled
with a fluorescent dye;
(P8) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 6 to 50 bases including the 155th to the 160th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 160th base is a cytosine labeled with a fluorescent dye;
(P8') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 6 to
50 bases including the 155th to the 160th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 160th base is a cytosine labeled
with a fluorescent dye;
(P9) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 12 to 50 bases including the 144th to the 155th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 144th base is a cytosine labeled with a fluorescent dye;
(P9') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 12 to
50 bases including the 144th to the 155th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 144th base is a cytosine labeled
with a fluorescent dye;
(P10) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 8 to 50 bases including the 148th to the 155th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 148th base is a cytosine labeled with a fluorescent dye;
(P10') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 8 to
50 bases including the 148th to the 155th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 148th base is a cytosine labeled
with a fluorescent dye;
(P11) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 5 to 50 bases including the 151st to the 155th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 151st base is a cytosine labeled with a fluorescent dye; or
(P11') an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 5 to
50 bases including the 151 st to the 155th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 151st base is a cytosine labeled
with a fluorescent dye.
The method of detecting a polymorphism in the PIK3CA gene according to the present
invention is a method which includes detecting a polymorphism in the PIK3CA gene using
at least one probe for detecting a polymorphism in the PIK3CA gene as described above.
The method of evaluating the efficacy of a drug according to the present invention
is a method which includes detecting a polymorphism in the PIK3CA gene by the above-described
method of detecting a polymorphism in the PIK3CA gene, and evaluating (e.g. determining
or predicting) the tolerance to a drug or the efficacy of a drug based on the detected
presence or absence of the polymorphism.
The reagent kit for detecting a polymorphism according to the present invention is
a kit which contains the probe for detecting a polymorphism in the PIK3CA gene.
[0015] The "PIK3CA gene" in the present invention is already known, and the base sequence
thereof corresponds to the 50001 st to the 91190th bases of NCBI Accession No. NG012113.
The PIK3CA gene encodes the PI3 kinase of the PI3 kinase-Akt signaling pathway which
plays an important role in regulating cell differentiation, development, growth and
maintenance.
[0016] The base sequence of SEQ ID NO:1 is a sequence of the 74541st to the 74960th bases
of the NCBI dbSNP Accession No. NG012113 and corresponds to a part of the base sequence
of the nucleic acid corresponding to the exon 9 of the PIK3CA gene.
The base sequence of SEQ ID NO:2 has the same base sequence as indicated in SEQ ID
NO:1 except that the base corresponding to the 232nd base, G (guanine), is mutated
to A (adenine) (hereinafter, also referred to as "G1624A").
The base sequence of SEQ ID NO:3 has the same base sequence as indicated in SEQ ID
NO:1 except that the base corresponding to the 24 1 st base, G, is mutated to A (hereinafter,
also referred to as "G1633A").
[0017] The base sequence of SEQ ID NO:4 is a sequence of the 90621st to the 90920th bases
of the NCBI dbSNP Accession No. NG012113 and corresponds to a part of the base sequence
of the nucleic acid corresponding to the exon 20 of the PIK3CA gene.
The base sequence of SEQ ID NO:5 has the same base sequence as indicated in SEQ ID
NO:4 except that the base corresponding to the 155th base, A, is mutated to G (hereinafter,
also referred to as "A3140G").
[0018] In the present invention, the description of the base sequences of the sample nucleic
acid to be detected in a sample and the polymorphism detection probe or primer shall
also apply to complementary base sequences thereof, respectively, unless otherwise
specified. Further, when the description of a particular base sequence is applied
to a complementary base sequence thereof, the descriptions of base sequences recognized
by the particular base sequence in the present invention should be applied provided
that the recognition by the particular base sequence is replaced with recognition
by a complementary base sequence of the particular base sequence, within a range of
the common general technical knowledge of those skilled in the art.
[0019] In the present invention, the term "Tm value" is defined as the temperature at which
a double-stranded nucleic acid dissociates (dissociation temperature: Tm), and is
generally defined as the temperature at which the absorbance at 260 nm has increased
by 50% of the total increase in absorbance resulting from complete dissociation of
the double-stranded nucleic acid. More specifically, when a solution containing a
double-stranded nucleic acid such as a double-stranded DNA is heated, the absorbance
at 260 nm of the double-stranded nucleic acid gradually increases. This is because
the hydrogen bonds between both strands of the double-stranded DNA are broken by heating,
thereby dissociating the double-stranded DNA into single-stranded DNA (melting of
DNA). When the double-stranded DNA has completely dissociated into single-stranded
DNA, the single-stranded DNA exhibit an absorbance that is about 1.5 times the absorbance
at the time of the initiation of the heating (i.e., the absorbance when the entire
DNA is in the form of a double-stranded DNA), which serves as an indicator of the
completion of the melting. The Tm value is defined based on this phenomenon.
In the present invention, when the phrase "the first to third bases from the 3' end"
is used in connection to an oligonucleotide sequence, it is assumed that the base
at the 3' end of the oligonucleotide chain is the first base from the 3' end. Similarly,
when the phrase "the first to third bases from the 5' end" is used in connection to
an oligonucleotide sequence, it is assumed that the base at the 5' end of the oligonucleotide
chain is the first base from the 5' end.
[0020] In the present specification, the scope of the term "process" or "step" includes
not only a discrete process/step, but also a process/step that cannot be clearly distinguished
from another process/step as long as the expected effect of the process/step of interest
is achieved.
In the present specification, any numerical range expressed using "to" refers to a
range including the numerical values before and after "to" as the minimum and maximum
values, respectively.
In the case in which the amount of a component that may be included in the composition
is indicated in the present invention, when there are multiple substances corresponding
to the component in the composition, the indicated amount means the total amount of
the multiple substances present in the composition, unless specifically stated otherwise.
In the present specification, the term "mutation" refers to a base sequence newly
produced by substitution, deletion, overlapping of or insertion into a part of a wild-type
base sequence. Further, the term "polymorphism" refers to a polymorphism of a base
sequence caused by a mutation.
The present invention is described below.
[0021] <Probe for Detecting Polymorphism in PIK3CA Gene>
The probe for detecting a polymorphism in the PIK3CA gene according to the present
invention (hereinafter, may be simply referred to as "the polymorphism detection probe")
is a probe for detecting a polymorphism in the PIK3CA gene which is at least one fluorescently
labeled oligonucleotide selected from the above-described P1 to P11'.
[0022] The above-described P1 to P11' fluorescently labeled oligonucleotides of the present
invention also encompass those fluorescently labeled oligonucleotides that are observed
to have homology to the sequence defined P1 to P11' fluorescently labeled oligonucleotides.
Specifically, in the present invention, a fluorescently labeled oligonucleotide observed
to have homology may be any fluorescently labeled oligonucleotide as long as it exhibits
an action comparable to that of the sequence defined P1 to P11' fluorescently labeled
oligonucleotides, and such a fluorescently labeled oligonucleotide preferably exhibits
an identity of not less than 80% to the sequence defined P1 to P11' oligonucleotides
or the corresponding portions of SEQ ID NOs: 1 to 5 or their complementary sequences.
From the standpoint of the detection sensitivity, such a fluorescently labeled oligonucleotide
may also exhibit an identity of not less than 85%, not less than 90%, not less than
95%, not less than 96%, not less than 97%, not less than 98% or not less than 99%.
When the fluorescently labeled oligonucleotide exhibits an identity of not less than
80%, there are advantages in that, for example, a high detection sensitivity is attained
for a sample nucleic acid containing a mutant-type PIK3CA gene.
[0023] Further, in the present invention, the above-described P1 to P11' fluorescently labeled
oligonucleotides also encompass those fluorescently labeled oligonucleotides which
hybridize, under stringent conditions, to a complementary strand of the sequence defined
P 1 to P11' fluorescently labeled oligonucleotides, as long as they exhibit an action
comparable to that of the sequence defined P1 to P11' fluorescently labeled oligonucleotides.
[0024] The hybridization may be carried out (under stringent conditions) according to a
known method or a method corresponding thereto, such as a method as described in
Molecular Cloning 3rd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001). This document is incorporated herein by reference.
The term "stringent conditions" means conditions in which specific hybrids are formed,
but non-specific hybrids are not formed. Typical examples of the stringent conditions
include, for example, conditions in which the hybridization is carried out at a potassium
concentration from about 25 mM (e.g. 25 mM) to about 50 mM (e.g. 50 mM) and a magnesium
concentration from about 1.0 mM (e.g. 1.0 mM) to about 5.0 mM (e.g. 5.0 mM). One example
of the conditions of the present invention is conditions in which the hybridization
is carried out in Tris-HCl (pH 8.6), 25 mM KC1, and 1.5 mM MgCl
2, but examples of the conditions of the present invention are not limited thereto.
Other examples of the stringent conditions are described in
Molecular Cloning 3rd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001). This document is incorporated herein by reference.
Those skilled in the art may readily choose such conditions by changing the hybridization
reaction and/or the salt concentration of the hybridization reaction solution.
[0025] Furthermore, the P1 to P11' fluorescently labeled oligonucleotides of the present
invention encompass a fluorescently labeled oligonucleotide having a sequence wherein
a base(s) has been inserted to, deleted from and/or substituted in P1 to P11' fluorescently
labeled oligonucleotides.
The fluorescently labeled oligonucleotide having a sequence wherein a base(s) has
been inserted, deleted and/or substituted is not particularly limited, as long as
the oligonucleotide exhibits an effect similar to that of P1 to P11' fluorescently
labeled oligonucleotides; and, in cases where a base(s) has been inserted, deleted
and/or substituted, the position(s) of the insertion(s), deletion(s) and/or substitution(s)
is not particularly limited. The number of bases that has been inserted, deleted and/or
substituted may be, for example, 1 base, or 2 or more bases, such as from 1 base to
10 bases and from 1 base to 5 bases, although varying depending on the total length
of the fluorescently labeled oligonucleotide.
[0026] The oligonucleotides in the above-described fluorescently labeled oligonucleotides
also encompass oligonucleotides as well as modified oligonucleotides.
Examples of a structural unit of the above-described oligonucleotide include ribonucleotides,
deoxyribonucleotides and artificial nucleic acids. Examples of the artificial nucleic
acids include DNAs, RNAs, LNAs (Locked Nucleic Acids) which are RNA analogues, PNAs
(Peptide Nucleic Acids) which are peptide nucleic acids, BNAs (Bridged Nucleic Acids)
which are cross-linked nucleic acids, and the like.
The above-described oligonucleotides may be constituted by one or multiple types of
the structural units described in the above.
[0027] In the above-described P1 to P11' fluorescently labeled oligonucleotides, a fluorescently
labeled base exists preferably at any one of the first to the third positions from
an end of the respective oligonucleotides, and more preferably at an end of the respective
oligonucleotides. By this, for example, the sensitivity for detecting a polymorphism
is further improved. In addition, the P1 to P11' fluorescently labeled oligonucleotides
may be obtained with good productivity.
[0028] The details of the fluorescently labeled oligonucleotides according to the present
invention will now be described in turn, starting with the P1 and P1' fluorescently
labeled oligonucleotides.
[0029] The above-described P1 and P1' fluorescently labeled oligonucleotides of the present
invention are probes capable of detecting a polymorphism of at least one base selected
from the group consisting of the 232nd to the 24 1 st bases of the base sequence indicated
in SEQ ID NO:1.
[0030] In the wild-type PIK3CA gene, the base corresponding to the 232nd base of the sequence
indicated in SEQ ID NO:1 is G (guanine); however, in a mutant-type, the G is mutated
to A (adenine) (hereinafter, also referred to as "C1624A"), and this base corresponds
to the 1624th base of the PIK3CA gene.
[0031] In the wild-type PIK3CA gene, the base corresponding to the 241 st base of the sequence
indicated in SEQ ID NO:1 is G (guanine); however, in a mutant-type, the G is mutated
to A (adenine) (hereinafter, also referred to as "G1633A"), and this base corresponds
to the 1633rd base of the PIK3CA gene.
[0032] The above-described P1 fluorescently labeled oligonucleotide of the present invention
has an identity of at least 80% to a base sequence having a length of 7 to 50 bases
including the 226th to the 232nd bases of the base sequence indicated in any one of
SEQ ID NOs: 1 to 3. Here, the base corresponding to the 226th base is a cytosine.
[0033] From the standpoint of detection sensitivity, the P1 fluorescently labeled oligonucleotide
of the present invention may also exhibit an identity of not less than 85%, not less
than 90%, not less than 95%, not less than 96%, not less than 97%, not less than 98%
or not less than 99% to a base sequence having a length of 7 to 50 bases including
the 226th to the 232nd bases of the base sequence indicated in any one of SEQ ID NOs:
1 to 3. As referred to herein, said 7 to 50 bases are consecutive bases of SEQ ID
NOs:1 to 3 which include the 226th to 232nd bases of the base sequence. Similar considerations
apply to the other oligonucleotides which are similarly defined. When the identity
is less than 80%, the detection sensitivity for a sample nucleic acid containing a
mutant-type PIK3CA gene becomes low.
[0034] Further, from the standpoint of detecting a polymorphism in the 241st base of the
base sequence indicated in SEQ ID NO:1, the form of the P1 fluorescently labeled oligonucleotide
also includes a base sequence having an identity of at least 80% to a base sequence
having a length of 16 to 50 bases including the 226th to the 24 1 st bases of the
base sequence indicated in any one of SEQ ID NOs:1 to 3.
From the standpoint of detection sensitivity, the P1 fluorescently labeled oligonucleotide
of the present invention may also exhibit an identity of not less than 85%, not less
than 90%, not less than 95%, not less than 96%, not less than 97%, not less than 98%
or not less than 99% to a base sequence having a length of 16 to 50 bases including
the 226th to the 241st bases of the base sequence indicated in any one of SEQ ID NOs:1
to 3. When the P1 fluorescently labeled oligonucleotide exhibits an identity of not
less than 80%, there are advantages in that, for example, a high detection sensitivity
is attained for a sample nucleic acid containing a mutant-type PIK3CA gene.
[0035] The above-described P1' fluorescently labeled oligonucleotide of the present invention
hybridizes, under stringent conditions, to the complementary strand of a base sequence
having a length of 7 to 50 bases including the 226th to the 232nd bases of the base
sequence indicated in any one of SEQ ID NOs:1 to 3. Here, the base corresponding to
the 226th base is a cytosine.
With regard to hybridization and stringent conditions, those matters described in
the above are applied. The same applies hereinafter.
[0036] Further, from the standpoint of detecting a polymorphism in the 241st base of the
base sequence indicated in SEQ ID NO:1, it is more preferred that the P1' fluorescently
labeled oligonucleotide take a form in which it hybridizes, under stringent conditions,
to the complementary strand of a base sequence having a length of 16 to 50 bases including
the 226th to the 241st bases of the base sequence indicated in any one of SEQ ID NOs:1
to 3.
[0037] The above-described P1 and P1' fluorescently labeled oligonucleotides of the present
invention are required to have a length of 7 mer to 50 mer. When the P1 and P1' fluorescently
labeled oligonucleotides have a length shorter than 7 mer or longer than 50 mer, the
sensitivity for detecting a polymorphism in the PIK3CA gene is decreased.
Further, the P1 and P1' fluorescently labeled oligonucleotides of the present invention
may have a length of 7 mer to 40 mer, 14 mer to 30 mer, or 16 mer to 25 mer. By setting
the length in the range of 10 mer to 40 mer, for example, the detection sensitivity
tends to be increased.
[0038] By changing the base lengths of the P1 and P1' fluorescently labeled oligonucleotides,
the Tm value, which is the dissociation temperature of a hybrid formed between the
fluorescently labeled oligonucleotides and their respective complementary strands
(target sequences), can be adjusted to a desired value.
[0039] Table 1 shows examples of the base sequences of the above-described P1 and P1' fluorescently
labeled oligonucleotides according to the present invention; however, the present
invention is not restricted thereto. Here, in Table 1, the capital letters indicate
the bases corresponding to the polymorphisms. The same applies hereinafter.
Further, in Table 1, the underlined cytosine represents the cytosine at the 226th
position of the base sequence indicated in any one of SEQ ID NOs:1 to 3.
[0040]
[Table 1]
|
|
|
SEQ ID NO: |
P1 |
5T-PIK3CA G1633A F1-19 |
(TAMRA)-ctctctGaaatcactAagc-P |
6 |
P1 |
5FL-PIK3CA-G1624A-F1 |
(BODIPY FL)-ctctctAaaatcactGagc-P |
7 |
[0041] Further, in the above-described P1 and P1' fluorescently labeled oligonucleotides
of the present invention, it is required that the 226th base (cytosine) is labeled
with a fluorescent dye.
[0042] The fluorescently labeled oligonucleotide of the present invention may be a fluorescently
labeled oligonucleotide in which the fluorescence intensity at the time when the oligonucleotide
is hybridized to its target sequence is decreased (quenched) or increased as compared
to the fluorescence intensity at the time when the oligonucleotide is not hybridized
to its target sequence. In particular, the fluorescently labeled oligonucleotide of
the present invention may be a fluorescently labeled oligonucleotide in which the
fluorescence intensity at the time when the oligonucleotide is hybridized to its target
sequence is decreased as compared to the fluorescence intensity at the time when the
oligonucleotide is not hybridized to its target sequence.
[0043] A probe that uses the "fluorescence quenching phenomenon" as described above is generally
referred to as a guanine quenching probe, and it is known as Q PROBE
®. Among such probes, an oligonucleotide which has been designed so that its 3' or
5' end is a cytosine (C) and which has been labeled with a fluorescent dye so that
the fluorescence emission is reduced when the C at the 3' or 5' end comes into proximity
with a guanine (G) is especially preferable. By using such a probe, the hybridization
and dissociation of the probe may be readily checked by the change in its signal.
[0044] A known detection method other than the detection method using a Q PROBE
® may also be applied. Examples of such a detection method include a TAQ-MAN probe
method, a hybridization probe method, a molecular beacon method, and a MGB probe method.
[0045] The fluorescent dye is not particularly limited, and examples of the fluorescent
dye include fluorescein, phosphor, rhodamine and polymethine dye derivatives. Examples
of commercially available products of such fluorescent dyes include Pacific Blue,
BODIPY FL, FluorePrime, Fluoredite, FAM, Cy3 and Cy5, and TAMRA.
The detection conditions of the fluorescently-labeled oligonucleotide are not particularly
limited, and may be decided, as appropriate, in accordance with the fluorescent dye
to be used. For example, Pacific Blue can be detected at a detection wavelength from
445 nm to 480 nm, TAMRA can be detected at a detection wavelength from 585 nm to 700
nm, and BODIPY FL can be detected at a detection wavelength from 520 nm to 555 nm.
By using a probe having such a fluorescent dye, hybridization and dissociation of
the probe can be readily confirmed based on the change in fluorescence signal therefrom.
Attachment of a fluorescent dye to the oligonucleotide may be carried out according
to an ordinary method, such as a method described in
JP-A No. 2002-119291.
It should be noted that, in the present invention, the same fluorescent dye may be
used, or alternatively, different fluorescent dyes may be used to label one or more
of the oligonucleotides.
[0046] In addition, the fluorescently-labeled oligonucleotide may have, for example, a phosphate
group added to its 3' end. Addition of a phosphate group to the 3' end of the fluorescently-labeled
oligonucleotide suppresses elongation of the probe itself by a gene amplification
reaction. As described below, DNA in which the presence or absence of a mutation should
be detected (target DNA) may be prepared using a gene amplification method such as
PCR. When the fluorescently-labeled oligonucleotide that has a phosphate group added
to its 3' end is used, the amplification reaction can be carried out even in the presence
of the oligonucleotide in the reaction solution of the amplification reaction.
A similar effect can be obtained also by adding a labeling substance (a fluorescent
dye) as described above to the 3' end.
[0047] The above-described P1 and P1' fluorescently labeled oligonucleotides may be used
as a probe for detecting a polymorphism in the PIK3CA gene, particularly at least
one of the polymorphisms selected from the group consisting of G1624A and G1633A.
In addition, the probe for detecting a polymorphism in the PIK3CA gene may be used
as a probe for melting curve analysis.
The P 1 and P1' fluorescently labeled oligonucleotide according to the present invention
may be produced according to a conventional method known as a method for synthesizing
an oligonucleotide, such as a method as described in
JP-ANo. 2002-119291, except that bases are used so that the base corresponding to the 226th base in the
base sequence indicated in SEQ ID NO:1 is a cytosine and the base corresponding to
the 226th base is labeled with a fluorescent dye.
[0048] More preferable embodiments of the P1 fluorescently labeled oligonucleotide and the
P1' fluorescently labeled oligonucleotide include the following P1-1 fluorescently
labeled oligonucleotide and the following P1'-1 fluorescently labeled oligonucleotide:
(P1-1) an oligonucleotide having an identity of at least 80% to a base sequence having
a length of 7 to 50 bases including the 226th to the 232nd bases of the base sequence
indicated in any one of SEQ ID NOs: 1 to 3, wherein the base corresponding to the
226th base is a cytosine labeled with a fluorescent dye, the oligonucleotide recognizing
a polymorphism of at least one base selected from the group consisting of the 232nd
and the 241 st bases of SEQ ID NO:1; and
(P1'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence having a length of 7 to 50 bases including the 226th to
the 232nd bases of the base sequence indicated in any one of SEQ ID NOs:1 to 3, wherein
the base corresponding to the 226th base is a cytosine labeled with a fluorescent
dye, the oligonucleotide recognizing a polymorphism of at least one base selected
from the group consisting of the 232nd and the 24 1 st bases of SEQ ID NO:1.
It should be noted that "recognizing a polymorphism of at least one base selected
from the group consisting of the 232nd and the 241 st bases of SEQ ID NO:1" has the
same meaning as "binding to at least one base selected from the group consisting of
the 232nd and the 241st bases of SEQ ID NO:1, which exhibits a polymorphism".
[0049] The above-described P2 to P11' fluorescently labeled oligonucleotides will now be
described; however, for those matters that are redundant due to the description provided
above in relation to the P1 and P1' fluorescently labeled oligonucleotides, the above
descriptions shall be referred to.
[0050] The above-described P2, P2', P3 and P3' fluorescently labeled oligonucleotides of
the present invention are probes capable of detecting a polymorphism at the 155th
base of the base sequence indicated in SEQ ID NO:4.
[0051] In the wild-type PIK3CA gene, the base corresponding to the 155th base of the sequence
indicated in SEQ ID NO:4 is A; however, in a mutant-type, the A is mutated to G (hereinafter,
also referred to as "A314OG"), and this base corresponds to the 3140th base of the
PIK3 CA gene.
[0052] The above-described P2 fluorescently labeled oligonucleotide of the present invention
has an identity of at least 80% to a base sequence having a length of 17 to 50 bases
including the 139th to the 155th bases of the base sequence indicated in SEQ ID NO:4
or 5. Here, the base corresponding to the 139th base is a cytosine. With regard to
the identity, the description provided in relation to the P1 fluorescently labeled
oligonucleotide shall be referred to.
The above-described P2' fluorescently labeled oligonucleotide of the present invention
hybridizes, under stringent conditions, to the complementary strand of a base sequence
having a length of 17 to 50 bases including the 139th to the 155th bases of the base
sequence indicated in SEQ ID NO:4 or 5. Here, the base corresponding to the 139th
base is a cytosine.
[0053] The above-described P3 fluorescently labeled oligonucleotide of the present invention
has an identity of at least 80% to a base sequence having a length of 11 to 50 bases
including the 155th to the 165th bases of the base sequence indicated in SEQ ID NO:4
or 5. Here, the base corresponding to the 165th base is a cytosine. With regard to
the identity, the description provided in relation to the P1 fluorescently labeled
oligonucleotide shall be referred to.
The above-described P3' fluorescently labeled oligonucleotide of the present invention
hybridizes, under stringent conditions, to the complementary strand of a base sequence
having a length of 11 to 50 bases including the 155th to the 165th bases of the base
sequence indicated in SEQ ID NO:4 or 5. Here, the base corresponding to the 165th
base is a cytosine.
[0054] The above-described P2 and P2' fluorescently labeled oligonucleotides of the present
invention are required to have a length of 17 mer to 50 mer. When the P2 and P2' fluorescently
labeled oligonucleotides have a length shorter than 17 mer or longer than 50 mer,
the sensitivity for detecting a polymorphism in the PIK3CA gene is decreased; therefore,
such a length is not preferred.
Further, the P2 and P2' fluorescently labeled oligonucleotides of the present invention
preferably have a length of 17 mer to 40 mer, more preferably 17 mer to 30 mer, still
more preferably 18 mer to 25 mer. By setting the length in the range of 17 mer to
40 mer, for example, the detection sensitivity tends to be increased.
[0055] The above-described P3 and P3' fluorescently labeled oligonucleotides of the present
invention are required to have a length of 11 mer to 50 mer. When the P3 and P3' fluorescently
labeled oligonucleotides have a length shorter than 11 mer or longer than 50 mer,
the sensitivity for detecting a polymorphism in the PIK3CA gene is decreased; therefore,
such a length is not preferred.
Further, the P3 and P3' fluorescently labeled oligonucleotides of the present invention
preferably have a length of 11 mer to 40 mer, more preferably 12 mer to 30 mer, still
more preferably 13 mer to 25 mer. By setting the length in the range of 11 mer to
40 mer, for example, the detection sensitivity tends to be increased.
[0056] By changing the base lengths of the P2, P2', P3 and P3' fluorescently labeled oligonucleotides,
the Tm value, which is the dissociation temperature of a hybrid formed between the
fluorescently labeled oligonucleotides and their respective complementary strands
(target sequences), can be adjusted to a desired value.
[0057] Table 2 shows examples of the base sequences of the above-described P2, P2', P3 and
P3' fluorescently labeled oligonucleotides according to the present invention; however,
the present invention is not restricted thereto.
Further, in Table 2, the underlined cytosine of SEQ ID NO:8 represents the cytosine
at the 139th position of the base sequence indicated in SEQ ID NO:4 or 5. Also, in
Table 2, the underlined cytosines of SEQ ID NOs:9 and 10 represent the cytosine at
the 165th position of the base sequence indicated in SEQ ID NO:4 or 5.
[0058]
[Table 2]
|
|
|
SEQ ID NO: |
P2 |
5T-PIK3CA3140-Wt-F3 |
(TAMRA)-caaatgaatgatgcacAtca-P |
8 |
P3 |
3T-PIK3CA3140-Wt-F8 |
atgcacAtcatggtggc-(TAMRA) |
9 |
P3 |
T-PIK3CA-3140-F10 |
atgcacAtcatggtgg(c-(TAMRA))t-P |
10 |
[0059] In the above-described P2 and P2' fluorescently labeled oligonucleotides of the present
invention, it is required that the 139th base (cytosine) is labeled with a fluorescent
dye.
In the above-described P3 and P3' fluorescently labeled oligonucleotides of the present
invention, it is required that the 165th base (cytosine) is labeled with a fluorescent
dye.
[0060] The above-described P2, P2', P3 and P3' fluorescently labeled oligonucleotides may
be used as a probe for detecting a polymorphism in the PIK3CA gene, particularly as
a probe for detecting a polymorphism in the PIK3CA gene which detects an A3140G mutation.
In addition, the probe for detecting a polymorphism in the PIK3CA gene may be used
as a probe for melting curve analysis.
[0061] More preferable embodiments of the P2 fluorescently labeled oligonucleotide and the
P2' fluorescently labeled oligonucleotide include the following P2-1 fluorescently
labeled oligonucleotide and the following P2'-1 fluorescently labeled oligonucleotide:
(P2-1) an oligonucleotide having an identity of at least 80% to a base sequence having
a length of 17 to 50 bases including the 139th to the 155th bases of the base sequence
indicated in SEQ ID NO:4 or 5, wherein the base corresponding to the 139th base is
a cytosine labeled with a fluorescent dye, the oligonucleotide recognizing a polymorphism
at the 155th base of SEQ ID NO:4; and
(P2'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence having a length of 17 to 50 bases including the 139th to
the 155th bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base
corresponding to the 139th base is a cytosine labeled with a fluorescent dye, the
oligonucleotide recognizing a polymorphism at the 155th base of SEQ ID NO:4.
[0062] More preferable embodiments of the P3 fluorescently labeled oligonucleotide and the
P3' fluorescently labeled oligonucleotide include the following P3-1 fluorescently
labeled oligonucleotide and the following P3'-1 fluorescently labeled oligonucleotide:
(P3-1) an oligonucleotide having an identity of at least 80% to a base sequence having
a length of 11 to 50 bases including the 155th to the 165th bases of the base sequence
indicated in SEQ ID NO:4 or 5, wherein the base corresponding to the 165th base is
a cytosine labeled with a fluorescent dye, the oligonucleotide recognizing a polymorphism
at the 155th base of SEQ ID NO:4; and
(P3'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence having a length of 11 to 50 bases including the 155th to
the 165th bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base
corresponding to the 165th base is a cytosine labeled with a fluorescent dye, the
oligonucleotide recognizing a polymorphism at the 155th base of SEQ ID NO:4.
[0063] The above-described P4 to P11' fluorescently labeled oligonucleotides of the present
invention are probes capable of detecting a polymorphism in a base sequence complementary
to the base sequence indicated in SEQ ID NO:4, particularly a polymorphism at the
155th base of the base sequence indicated in SEQ ID NO:4.
[0064] The above-described P4 fluorescently labeled oligonucleotide of the present invention
has an identity of at least 80% to a base sequence complementary to a base sequence
having a length of 13 to 50 bases including the 155th to the 167th bases of the base
sequence indicated in SEQ ID NO:4 or 5. Here, the base corresponding to the 167th
base is required to be a cytosine. With regard to the identity, those descriptions
provided in relation to the P1 fluorescently labeled oligonucleotide shall be referred
to. The same applies hereinafter.
The above-described P4' fluorescently labeled oligonucleotide of the present invention
hybridizes, under stringent conditions, to the complementary strand of a base sequence
complementary to a base sequence having a length of 13 to 50 bases including the 155th
to the 167th bases of the base sequence indicated in SEQ ID NO:4 or 5. In other words,
the P4' fluorescently labeled oligonucleotide of the present invention hybridizes,
under stringent conditions, to a base sequence having a length of 13 to 50 bases including
the 155th to the 167th bases of the base sequence indicated in SEQ ID NO:4 or 5. Here,
the base corresponding to the 167th base is a cytosine.
[0065] The above-described P5 fluorescently labeled oligonucleotide of the present invention
has an identity of at least 80% to a base sequence complementary to a base sequence
having a length of 10 to 50 bases including the 155th to the 164th bases of the base
sequence indicated in SEQ ID NO:4 or 5. Here, the base corresponding to the 164th
base is a cytosine.
The above-described P5' fluorescently labeled oligonucleotide of the present invention
hybridizes, under stringent conditions, to the complementary strand of a base sequence
complementary to a base sequence having a length of 10 to 50 bases including the 155th
to the 164th bases of the base sequence indicated in SEQ ID NO:4 or 5. In other words,
the P5' fluorescently labeled oligonucleotide of the present invention hybridizes,
under stringent conditions, to a base sequence having a length of 10 to 50 bases including
the 155th to the 164th bases of the base sequence indicated in SEQ ID NO:4 or 5. Here,
the base corresponding to the 164th base is a cytosine.
[0066] The above-described P6 fluorescently labeled oligonucleotide of the present invention
has an identity of at least 80% to a base sequence complementary to a base sequence
having a length of 9 to 50 bases including the 155th to the 163rd bases of the base
sequence indicated in SEQ ID NO:4 or 5. Here, the base corresponding to the 163rd
base is a cytosine.
The above-described P6' fluorescently labeled oligonucleotide of the present invention
hybridizes, under stringent conditions, to the complementary strand of a base sequence
complementary to a base sequence having a length of 9 to 50 bases including the 155th
to the 163rd bases of the base sequence indicated in SEQ ID NO:4 or 5. In other words,
the P6' fluorescently labeled oligonucleotide of the present invention hybridizes,
under stringent conditions, to a base sequence having a length of 9 to 50 bases including
the 155th to the 163rd bases of the base sequence indicated in SEQ ID NO:4 or 5. Here,
the base corresponding to the 163rd base is a cytosine.
[0067] The above-described P7 fluorescently labeled oligonucleotide of the present invention
has an identity of at least 80% to a base sequence complementary to a base sequence
having a length of 7 to 50 bases including the 155th to the 161st bases of the base
sequence indicated in SEQ ID NO:4 or 5. Here, the base corresponding to the 161st
base is a cytosine.
The above-described P7' fluorescently labeled oligonucleotide of the present invention
hybridizes, under stringent conditions, to the complementary strand of a base sequence
complementary to a base sequence having a length of 7 to 50 bases including the 155th
to the 161st bases of the base sequence indicated in SEQ ID NO:4 or 5. In other words,
the P7' fluorescently labeled oligonucleotide of the present invention hybridizes,
under stringent conditions, to a base sequence having a length of 7 to 50 bases including
the 155th to the 161st bases of the base sequence indicated in SEQ ID NO:4 or 5. Here,
the base corresponding to the 161 st base is a cytosine.
[0068] The above-described P8 fluorescently labeled oligonucleotide of the present invention
has an identity of at least 80% to a base sequence complementary to a base sequence
having a length of 6 to 50 bases including the 155th to the 160th bases of the base
sequence indicated in SEQ ID NO:4 or 5. Here, the base corresponding to the 160th
base is a cytosine.
The above-described P8' fluorescently labeled oligonucleotide of the present invention
hybridizes, under stringent conditions, to the complementary strand of a base sequence
complementary to a base sequence having a length of 6 to 50 bases including the 155th
to the 160th bases of the base sequence indicated in SEQ ID NO:4 or 5. In other words,
the P8' fluorescently labeled oligonucleotide of the present invention hybridizes,
under stringent conditions, to a base sequence having a length of 6 to 50 bases including
the 155th to the 160th bases of the base sequence indicated in SEQ ID NO:4 or 5. Here,
the base corresponding to the 160th base is a cytosine.
[0069] The above-described P9 fluorescently labeled oligonucleotide of the present invention
has an identity of at least 80% to a base sequence complementary to a base sequence
having a length of 12 to 50 bases including the 144th to the 155th bases of the base
sequence indicated in SEQ ID NO:4 or 5. Here, the base corresponding to the 144th
base is a cytosine.
The above-described P9' fluorescently labeled oligonucleotide of the present invention
hybridizes, under stringent conditions, to the complementary strand of a base sequence
complementary to a base sequence having a length of 12 to 50 bases including the 144th
to the 155th bases of the base sequence indicated in SEQ ID NO:4 or 5. In other words,
the P9' fluorescently labeled oligonucleotide of the present invention hybridizes,
under stringent conditions, to a base sequence having a length of 12 to 50 bases including
the 144th to the 155th bases of the base sequence indicated in SEQ ID NO:4 or 5. Here,
the base corresponding to the 144th base is a cytosine.
[0070] The above-described P10 fluorescently labeled oligonucleotide of the present invention
has an identity of at least 80% to a base sequence complementary to a base sequence
having a length of 8 to 50 bases including the 148th to the 155th bases of the base
sequence indicated in SEQ ID NO:4 or 5. Here, the base corresponding to the 148th
base is a cytosine.
The above-described P10' fluorescently labeled oligonucleotide of the present invention
hybridizes, under stringent conditions, to the complementary strand of a base sequence
complementary to a base sequence having a length of 8 to 50 bases including the 148th
to the 155th bases of the base sequence indicated in SEQ ID NO:4 or 5. In other words,
the P10' fluorescently labeled oligonucleotide of the present invention hybridizes,
under stringent conditions, to a base sequence having a length of 8 to 50 bases including
the 148th to the 155th bases of the base sequence indicated in SEQ ID NO:4 or 5. Here,
the base corresponding to the 148th base is a cytosine.
[0071] The above-described P11 fluorescently labeled oligonucleotide of the present invention
has an identity of at least 80% to a base sequence complementary to a base sequence
having a length of 5 to 50 bases including the 151 st to the 155th bases of the base
sequence indicated in SEQ ID NO:4 or 5. Here, the base corresponding to the 151st
base is a cytosine.
The above-described P11' fluorescently labeled oligonucleotide of the present invention
hybridizes, under stringent conditions, to the complementary strand of a base sequence
complementary to a base sequence having a length of 5 to 50 bases including the 151st
to the 155th bases of the base sequence indicated in SEQ ID NO:4 or 5. In other words,
the P11' fluorescently labeled oligonucleotide of the present invention hybridizes,
under stringent conditions, to a base sequence having a length of 5 to 50 bases including
the 151st to the 155th bases of the base sequence indicated in SEQ ID NO:4 or 5. Here,
the base corresponding to the 151 st base is a cytosine.
[0072] The above-described P4 and P4' fluorescently labeled oligonucleotides of the present
invention are required to have a length of 13 mer to 50 mer. When the P4 and P4' fluorescently
labeled oligonucleotides have a length shorter than 13 mer or longer than 50 mer,
the sensitivity for detecting a polymorphism in the PIK3CA gene is decreased; therefore,
such a length is not preferred.
Further, the P4 and P4' fluorescently labeled oligonucleotides of the present invention
preferably have a length of 13 mer to 40 mer, more preferably 13 mer to 30 mer, still
more preferably 14 mer to 25 mer. By setting the length in the range of 13 mer to
40 mer, for example, the detection sensitivity tends to be increased.
[0073] The above-described P5 and P5' fluorescently labeled oligonucleotides of the present
invention are required to have a length of 10 mer to 50 mer. When the P5 and P5' fluorescently
labeled oligonucleotides have a length shorter than 10 mer or longer than 50 mer,
the sensitivity for detecting a polymorphism in the PIK3CA gene is decreased; therefore,
such a length is not preferred.
Further, the P5 and P5' fluorescently labeled oligonucleotides of the present invention
preferably have a length of 10 mer to 40 mer, more preferably 12 mer to 30 mer, still
more preferably 13 mer to 25 mer. By setting the length in the range of 10 mer to
40 mer, for example, the detection sensitivity tends to be increased.
[0074] The above-described P6 and P6' fluorescently labeled oligonucleotides of the present
invention are required to have a length of 9 mer to 50 mer. When the P6 and P6' fluorescently
labeled oligonucleotides have a length shorter than 9 mer or longer than 50 mer, the
sensitivity for detecting a polymorphism in the PIK3CA gene is decreased; therefore,
such a length is not preferred.
Further, the P6 and P6' fluorescently labeled oligonucleotides of the present invention
preferably have a length of 9 mer to 40 mer, more preferably 11 mer to 30 mer, still
more preferably 13 mer to 25 mer. By setting the length in the range of 9 mer to 40
mer, for example, the detection sensitivity tends to be increased.
[0075] The above-described P7 and P7' fluorescently labeled oligonucleotides of the present
invention are required to have a length of 7 mer to 50 mer. When the P7 and P7' fluorescently
labeled oligonucleotides have a length shorter than 7 mer or longer than 50 mer, the
sensitivity for detecting a polymorphism in the PIK3CA gene is decreased; therefore,
such a length is not preferred.
Further, the P7 and P7' fluorescently labeled oligonucleotides of the present invention
preferably have a length of 7 mer to 40 mer, more preferably 10 mer to 30 mer, still
more preferably 13 mer to 25 mer. By setting the length in the range of 7 mer to 40
mer, for example, the detection sensitivity tends to be increased.
[0076] The above-described P8 and P8' fluorescently labeled oligonucleotides of the present
invention are required to have a length of 6 mer to 50 mer. When the P8 and P8' fluorescently
labeled oligonucleotides have a length shorter than 6 mer or longer than 50 mer, the
sensitivity for detecting a polymorphism in the PIK3CA gene is decreased; therefore,
such a length is not preferred.
Further, the P8 and P8' fluorescently labeled oligonucleotides of the present invention
preferably have a length of 7 mer to 40 mer, more preferably 10 mer to 30 mer, still
more preferably 13 mer to 25 mer. By setting the length in the range of 7 mer to 40
mer, for example, the detection sensitivity tends to be increased.
[0077] The above-described P9 and P9' fluorescently labeled oligonucleotides of the present
invention are required to have a length of 12 mer to 50 mer. When the P9 and P9' fluorescently
labeled oligonucleotides have a length shorter than 12 mer or longer than 50 mer,
the sensitivity for detecting a polymorphism in the PIK3CA gene is decreased; therefore,
such a length is not preferred.
Further, the P9 and P9' fluorescently labeled oligonucleotides of the present invention
preferably have a length of 12 mer to 40 mer, more preferably 12 mer to 30 mer, still
more preferably 13 mer to 25 mer. By setting the length in the range of 12 mer to
40 mer, for example, the detection sensitivity tends to be increased.
[0078] The above-described P10 and P10' fluorescently labeled oligonucleotides of the present
invention are required to have a length of 8 mer to 50 mer. When the P10 and P10'
fluorescently labeled oligonucleotides have a length shorter than 8 mer or longer
than 50 mer, the sensitivity for detecting a polymorphism in the PIK3CA gene is decreased;
therefore, such a length is not preferred.
Further, the P10 and P10' fluorescently labeled oligonucleotides of the present invention
preferably have a length of 8 mer to 40 mer, more preferably 11 mer to 30 mer, still
more preferably 13 mer to 25 mer. By setting the length in the range of 8 mer to 40
mer, for example, the detection sensitivity tends to be increased.
[0079] The above-described P11 and P11' fluorescently labeled oligonucleotides of the present
invention are required to have a length of 5 mer to 50 mer. When the P11 and P11'
fluorescently labeled oligonucleotides have a length shorter than 5 mer or longer
than 50 mer, the sensitivity for detecting a polymorphism in the PIK3CA gene is decreased;
therefore, such a length is not preferred.
Further, the P1 1 and P11' fluorescently labeled oligonucleotides of the present invention
preferably have a length of 7 mer to 40 mer, more preferably 10 mer to 30 mer, still
more preferably 13 mer to 25 mer. By setting the length in the range of 7 mer to 40
mer, for example, the detection sensitivity tends to be increased.
[0080] By changing the base lengths of the P4 to P11' fluorescently labeled oligonucleotides,
the Tm value, which is the dissociation temperature of a hybrid formed between the
fluorescently labeled oligonucleotides and their respective target sequences, can
be adjusted to a desired value.
[0081] In the present invention, the difference between the Tm value when each of the P1
to P11' fluorescently labeled oligonucleotides is hybridized with a wild-type sample
nucleic acid and the Tm value when each of the P1 to P11' fluorescently labeled oligonucleotides
is hybridized with a mutant-type sample nucleic acid is preferably not less than 1.5°C,
more preferably not less than 2.0°C, still more preferably not less than 5.0°C, and
yet still more preferably not less than 9.0°C.
[0082] Examples of a method of increasing the above-described difference in the Tm value
include a method by which a probe is allowed to contain a base which mismatches with
a base sequence corresponding to a region to which the probe hybridizes. Specific
examples include those methods described in
Nature Biotech (1997) vol. 15, pp. 331-335 and the like.
[0083] Table 3 shows examples of the base sequence of the above-described P4 to P11' fluorescently
labeled oligonucleotides according to the present invention; however, the present
invention is not restricted thereto.
Further, in Table 3, the underlined cytosines each represent the cytosine corresponding
to the following base position of the base sequence indicated in SEQ ID NO:4 or 5:
the 167th base for SEQ ID NO:11; the 164th base for SEQ ID NO:12; the 163rd base for
SEQ ID NO:13; the 161st base for SEQ ID NOs: 14 and 15; the 160th base for SEQ ID
NO:16; the 144th base for SEQ ID NO:17; the 148th base for SEQ ID NO:18; the 151st
base for SEQ ID NO:19; the 144th and the 161st bases for SEQ ID NO:20; the 148th and
the 164th bases for SEQ ID NO:21; and the 163rd base for SEQ ID NO:22.
[0084]
[Table 3]
|
|
|
SEQ ID NO: |
P4 |
5T-PIK3CA3140-Mt-R9 |
(TAMRA)-cagccaccatgaCgtgc-P |
11 |
P5 |
5T-PIK3CA3140-Mt-R10 |
(TAMRA)-ccaccatgaCgtgcatca-P |
12 |
P6 |
5T-PIK3CA3140-Mt-R11 |
(TAMRA)-caccatgaCgtgcatca-P |
13 |
P7 |
ST-PIK3CA A3140G R1-17 |
(TAMRA)-ccatgaCgtgcatcatt-P |
14 |
P7 |
5PB-PIC3CA A3140G R1 |
(Pacific Blue)-ccatgaCgtgcatcatt-P |
15 |
P8 |
5T-PIK3CA3140-Mt-R12 |
(TAMRA)-catgaCgtgcatcattcat-P |
16 |
P9 |
3T-PIK3CA3140-Mt-R16 |
accatgaCgtgcatcattc-(TAMRA) |
17 |
P10 |
3T-PIK3CA3140-Mt-R17 |
ccaccatgaCgtgcatc-(TAMRA) |
18 |
P11 |
3T-PIK3CA3140-Mt-R18 |
cagccaccatgaCgtgc-(TAMRA) |
19 |
P7-P9 |
35T-PIK3CA-3140-R19 |
(TAMRA)-ccatgaCgtgcatcattc-(TAMRA) |
20 |
P5-P11 |
35T-PIK3CA-3140-R20 |
(TAMRA)-ccaccatgaCgtgcatc-(TAMRA) |
21 |
P6 |
T-PIK3 CA-3140-R21 |
c(c-(TAMRA))atgaCgtgcatcattc-P |
22 |
[0085] In the above-described P4 and P4' fluorescently labeled oligonucleotides of the present
invention, it is required that the 167th base (cytosine) is labeled with a fluorescent
dye.
In the above-described P5 and P5' fluorescently labeled oligonucleotides of the present
invention, it is required that the 164th base (cytosine) is labeled with a fluorescent
dye.
In the above-described P6 and P6' fluorescently labeled oligonucleotides of the present
invention, it is required that the 163rd base (cytosine) is labeled with a fluorescent
dye.
In the above-described P7 and P7' fluorescently labeled oligonucleotides of the present
invention, it is required that the 161 st base (cytosine) is labeled with a fluorescent
dye.
In the above-described P8 and P8' fluorescently labeled oligonucleotides of the present
invention, it is required that the 160th base (cytosine) is labeled with a fluorescent
dye.
In the above-described P9 and P9' fluorescently labeled oligonucleotides of the present
invention, it is required that the 144th base (cytosine) is labeled with a fluorescent
dye.
In the above-described P10 and P10' fluorescently labeled oligonucleotides of the
present invention, it is required that the 148th base (cytosine) is labeled with a
fluorescent dye.
In the above-described P11 and P1 1' fluorescently labeled oligonucleotides of the
present invention, it is required that the 151 st base (cytosine) is labeled with
a fluorescent dye.
The above-described P4 to P11' fluorescently labeled oligonucleotides may be used
as a probe for detecting a polymorphism in the PIK3CA gene, particularly as a probe
for detecting a polymorphism in the PIK3CA gene which detects an A3140G mutation.
In addition, the probe for detecting a polymorphism in the PIK3CA gene may be used
as a probe for performing melting curve analysis.
[0086] More preferable embodiments of the P4 fluorescently labeled oligonucleotide and the
P4' fluorescently labeled oligonucleotide include the following P4-1 fluorescently
labeled oligonucleotide and the following P4'-1 fluorescently labeled oligonucleotide:
(P4-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 13 to 50 bases including the 155th to the 167th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 167th base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4; and (P4'-1) an oligonucleotide
which hybridizes under stringent conditions to the complementary strand of a base
sequence complementary to a base sequence having a length of 13 to 50 bases including
the 155th to the 167th bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein
the base corresponding to the 167th base is a cytosine labeled with a fluorescent
dye, the oligonucleotide recognizing a polymorphism at the 155th base of SEQ ID NO:4.
[0087] More preferable embodiments of the P5 fluorescently labeled oligonucleotide and the
P5' fluorescently labeled oligonucleotide include the following P5-1 fluorescently
labeled oligonucleotide and the following P5'-1 fluorescently labeled oligonucleotide:
(P5-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 10 to 50 bases including the 155th to the 164th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 164th base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4; and
(P5'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 10 to
50 bases including the 155th to the 164th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 164th base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4.
[0088] More preferable embodiments of the P6 fluorescently labeled oligonucleotide and the
P6' fluorescently labeled oligonucleotide include the following P6-1 fluorescently
labeled oligonucleotide and the following P6'-1 fluorescently labeled oligonucleotide:
(P6-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 9 to 50 bases including the 155th to the 163rd
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 163rd base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4; and
(P6'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 9 to
50 bases including the 155th to the 163rd bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 163rd base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4.
[0089] More preferable embodiments of the P7 fluorescently labeled oligonucleotide and the
P7' fluorescently labeled oligonucleotide include the following P7-1 fluorescently
labeled oligonucleotide and the following P7'-1 fluorescently labeled oligonucleotide:
(P7-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 7 to 50 bases including the 155th to the 161st
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 161 st base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4; and
(P7'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 7 to
50 bases including the 155th to the 161st bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 161st base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4.
[0090] More preferable embodiments of the P8 fluorescently labeled oligonucleotide and the
P8' fluorescently labeled oligonucleotide include the following P8-1 fluorescently
labeled oligonucleotide and the following P8'-1 fluorescently labeled oligonucleotide:
(P8-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 6 to 50 bases including the 155th to the 160th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 160th base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4; and
(P8'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 6 to
50 bases including the 155th to the 160th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 160th base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4.
[0091] More preferable embodiments of the P9 fluorescently labeled oligonucleotide and the
P9' fluorescently labeled oligonucleotide include the following P9-1 fluorescently
labeled oligonucleotide and the following P9'-1 fluorescently labeled oligonucleotide:
(P9-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 12 to 50 bases including the 144th to the 155th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 144th base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4; and
(P9'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 12 to
50 bases including the 144th to the 155th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 144th base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4.
[0092] More preferable embodiments of the P10 fluorescently labeled oligonucleotide and
the P10' fluorescently labeled oligonucleotide include the following P10-1 fluorescently
labeled oligonucleotide and the following P10'-1 fluorescently labeled oligonucleotide:
(P10-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 8 to 50 bases including the 148th to the 155th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 148th base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4; and
(P10'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 8 to
50 bases including the 148th to the 155th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 148th base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4.
[0093] More preferable embodiments of the P11 fluorescently labeled oligonucleotide and
the P11' fluorescently labeled oligonucleotide include the following P11-1 fluorescently
labeled oligonucleotide and the following P11'-1 fluorescently labeled oligonucleotide:
(P11-1) an oligonucleotide having an identity of at least 80% to a base sequence complementary
to a base sequence having a length of 5 to 50 bases including the 151st to the 155th
bases of the base sequence indicated in SEQ ID NO:4 or 5, wherein the base corresponding
to the 151 st base is a cytosine labeled with a fluorescent dye, the oligonucleotide
recognizing a polymorphism at the 155th base of SEQ ID NO:4; and
(P11'-1) an oligonucleotide which hybridizes under stringent conditions to the complementary
strand of a base sequence complementary to a base sequence having a length of 5 to
50 bases including the 151 st to the 155th bases of the base sequence indicated in
SEQ ID NO:4 or 5, wherein the base corresponding to the 151st base is a cytosine labeled
with a fluorescent dye, the oligonucleotide recognizing a polymorphism at the 155th
base of SEQ ID NO:4.
[0094] <Primer>
In the below-described method of detecting a polymorphism in the PIK3 CA gene, primers
are used in amplifying a sequence having a PIK3CA gene polymorphism to be detected
by a PCR method.
The primers that may be used in the present invention are not particularly restricted
as long as they are capable of amplifying a nucleic acid containing a base exhibiting
at least one polymorphism selected from the group consisting of G1624A mutation, G1633A
mutation and A3140G mutation, which are the target PIK3CA gene polymorphisms to be
detected.
[0095] The primer to be applied to the PCR method is not particularly limited, as long as
it is capable of amplifying a region to which the probe of the present invention may
be hybridized. Such a primer may be designed based on the base sequences indicated
in SEQ ID NO:1 to SEQ ID NO:5 by those skilled in the art. The length and Tm value
of the primer may be a length from 12 mer to 40 mer and a value from 40°C to 70°C,
or a length from 16 mer to 30 mer and a value from 55°C to 60°C.
The lengths of individual primers in a primer set do not need to be the same, although
the Tm values of these primers are preferably approximately the same (or the difference
between the Tm values of these primers is preferably within 5°C).
[0096] Examples of the primers that may be used for amplifying a base sequence containing
a region to which the polymorphism detection probe according to the present invention
used in the method of detecting a polymorphism according to the present invention
hybridizes are shown below. It is noted here that the following examples are provided
for illustrative purposes only and that, therefore, the present invention is not restricted
thereto. In Table 4, Y represents C or T.
[0097]
[Table 4]
|
Sequence (5'→3') |
mer |
SEQ ID NO: |
PIK3CA ex9 F |
gaacagctcaaagcaatttctacacgag |
28 |
23 |
PIK3CA ex9 R |
cagagaatYtccattttagcacttacYtgtgac |
33 |
24 |
PIK3CA A3140G F |
gaggctttggagtatttcatgaaacaaatg |
30 |
25 |
PIK3CA A3140G R |
gcatgctgtttaattgtgtggaagatccaatc |
32 |
26 |
[0098] When amplifying a base sequence containing a region to which the polymorphism detection
probe according to the present invention hybridizes, from the standpoint of sensitivity
and efficiency, for example, among the oligonucleotides shown in Table 4, the PIK3CA
ex9 F and the PIK3CA ex9 R, or the PIK3CAA3140G F and the PIK3CAA3140G R may be used
as a set of paired primers.
[0099] The method of detecting a polymorphism is not particularly limited, as long as it
is a method in which a fluorescently labeled oligonucleotide as described above is
used as a probe. As an example of the polymorphism detection method in which a fluorescently
labeled oligonucleotide as described above is used as a probe, a method of detecting
a polymorphism using Tm analysis is described below.
[0100] <Polymorphism Detection Method>
The method of detecting a polymorphism in the PIK3CA gene according to the present
invention is a method of detecting a polymorphism in the PIK3CA gene which includes
detecting a polymorphism in the PIK3CA gene by using at least one probe for detecting
a polymorphism in the PIK3CA gene as described above.
The method of detecting a polymorphism of the present invention may include at least
one probe for detecting a polymorphism as described above, and this may make it possible
to detect a polymorphism(s) in the PIK3CA gene easily and with high sensitivity.
In addition, the method of detecting a polymorphism according to the present invention
may be employed as a method of detecting a polymorphism in the PIK3CA gene, and may
include the below-described processes or steps (I) to (IV), and may include the below-described
step (V). The method of detecting a polymorphism according to the present invention
has the feature of using the above-described probe, and other configurations, conditions
and the like are not particularly limited by the description below.
[0101] Step (I): contacting the fluorescently-labeled probe with a single-stranded nucleic
acid in a sample, to obtain a hybrid.
Step (II): dissociating the hybrid by changing the temperature of the sample containing
the hybrid, and measuring the change in fluorescence signal due to the dissociation
of the hybrid.
Step (III): measuring the Tm value, which is the dissociation temperature of the hybrid,
based on the change in fluorescence signal.
Step (IV): detecting the presence of a polymorphism in the PIK3CA gene on the single-stranded
nucleic acid in the sample, based on the Tm value.
Step (V): determining the abundance ratio of the single-stranded nucleic acid having
the polymorphism in the total single-stranded nucleic acids contained in the sample,
based on the presence of the polymorphism(s).
[0102] Furthermore, the method according to the present invention may further include amplifying
the nucleic acid before obtaining the hybrid in step (I) or simultaneously with obtaining
the hybrid in step (I), in addition to steps (I) to (IV) or in addition to steps (I)
to (V).
The measurement of the Tm value in step (III) may include not only measuring the dissociation
temperature of the hybrid, but also measuring the differential values of the fluorescence
signal that changes according to the temperature when the hybrid is melted.
[0103] In the present invention, the nucleic acid in the sample may be a single-stranded
nucleic acid or a double-stranded nucleic acid. In the case in which the nucleic acid
is a double-stranded nucleic acid, the method may include, for example, melting (dissociating)
the double-stranded nucleic acid in the sample into single-stranded nucleic acids
by heating before being hybridized with the fluorescently-labeled probe. The dissociation
of a double-stranded nucleic acid into single-stranded nucleic acids enables hybridization
with the fluorescently-labeled probe.
[0104] In the present invention, the nucleic acid contained in the sample to be detected
may be, for example, a nucleic acid originally contained in a biological sample, or
an amplification product obtained by amplifying a region of the gene of interest that
contains a mutated site(s) of the PIK3CA gene by PCR or the like using a nucleic acid
originally contained in a biological sample as a template with a view to improving
the detection accuracy. The length of the amplification product is not particularly
limited, and may be, for example, a length from 50 mer to 1000 mer, or a length from
80 mer to 200 mer. Furthermore, the nucleic acid in the sample may be, for example,
a cDNA that has been synthesized from RNAs derived from a biological sample (e.g.,
total RNAs, mRNAs, etc.) by RT-PCR (Reverse Transcription PCR).
[0105] In the present invention, the addition ratio (molar ratio) of the probe according
to the present invention relative to the nucleic acids in the sample is not particularly
limited. The amount of the probe to be added may be, for example, no more than 1-fold
(by mol) of the amount of DNA in the sample. From the viewpoint of ensuring a sufficient
detection signal, the addition ratio of the probe according to the present invention
to be added relative to the nucleic acids in the sample (in a molar ratio) may be
0.1 or lower.
The "nucleic acids in the sample" may be, for example, the total nucleic acids to
be detected that have the polymorphism to be detected and nucleic acids, other than
the nucleic acids to be detected, that do not have the polymorphism, or the total
amplification products containing a detection target sequence having the polymorphism
to be detected and amplification products containing a sequence, other than the detection
target sequence, that does not have the polymorphism. Although the ratio of the nucleic
acid to be detected relative to nucleic acids in the sample is usually unknown in
advance, the consequent addition ratio of the probe relative to the nucleic acids
to be detected (or the amplification products containing a sequence to be detected)
(in a molar ratio) may be 10 or lower. The addition ratio of the probe relative to
the nucleic acids to be detected (or the amplification products containing a sequence
to be detected) (in a molar ratio) may be 5 or lower, or 3 or lower. The lower limit
of the ratio is not particularly limited, and may be, for example, 0.001 or higher,
0.01 or higher, or 0.1 or higher.
[0106] The above-described addition ratio of the fluorescently-labeled probe according to
the present invention relative to the DNA may be, for example, a molar ratio relative
to the double-stranded nucleic acids or a molar ratio relative to the single-stranded
nucleic acids.
[0107] In the present invention, the measurement of the change in the signal caused by a
temperature change for determining a Tm value may be carried out by measuring the
absorbance at 260 nm on the basis of the principle described above. However, the measurement
may be carried out by measuring a signal which is based on a signal from the label
attached to the fluorescently-labeled probe, and which varies in accordance with the
degree of formation of a hybrid of a single-stranded DNA and the probe. Therefore,
the above-described fluorescently-labeled oligonucleotide may be used as the fluorescently-labeled
probe. Examples of the fluorescently-labeled oligonucleotide (hereinafter sometimes
collectively referred to as "fluorescently-labeled oligonucleotide") include a fluorescently-labeled
oligonucleotide in respect of which the fluorescence intensity when the oligonucleotide
is hybridized with a target sequence thereof is decreased (quenched) as compared to
the fluorescence intensity when the oligonucleotide is not hybridized with the target
sequence thereof, and a fluorescently-labeled oligonucleotide in respect of which
the fluorescence intensity when the oligonucleotide is hybridized with a target sequence
thereof is increased as compared to the fluorescence intensity when the oligonucleotide
is not hybridized with the target sequence thereof.
The former fluorescently-labeled oligonucleotide does not show a fluorescence signal
or only a weak fluorescence signal when the fluorescently-labeled oligonucleotide
forms a hybrid (a double-stranded DNA) with the sequence to be detected; however,
the fluorescently-labeled oligonucleotide shows a fluorescence signal or shows an
increased fluorescence signal when the fluorescently-labeled oligonucleotide is dissociated
by heating.
The latter fluorescently-labeled oligonucleotide shows a fluorescence signal when
the fluorescently-labeled oligonucleotide forms a hybrid (a double-stranded DNA) with
the sequence to be detected; however, the fluorescently-labeled oligonucleotide shows
a decreased fluorescence signal or ceases to show a fluorescent signal when the fluorescently-labeled
oligonucleotide is dissociated by heating. Therefore, similar to the measurement of
the absorbance at 260 nm described above, the progress of melting can be monitored,
and the Tm value can be determined by detecting the change in fluorescence signal
from the fluorescent label under the conditions specific to the fluorescent label
(for example, the fluorescence wavelength thereof).
[0108] The method for detecting the change in the signal based on a signal from the fluorescent
dye in the polymorphism detection method according to the present invention is described
below by way of specific examples. The polymorphism detection method according to
the present invention has as a feature the use of the fluorescently-labeled polymorphism
detection probe, and other processes and conditions of the method are not limited
in any way.
[0109] The sample containing a nucleic acid that serves as a template for nucleic acid amplification
is not particularly limited as long as the sample contains a nucleic acid, particularly
the PIK3CA gene. Examples of such a sample include a sample that is derived from or
can be derived from any biological source, examples of which include: a tissue such
as colon or lung; a hemocyte such as a leukocyte cell; whole blood; plasma; sputum;
a suspension of oral mucosa; a somatic cell of nail, hair or the like; a germ cell;
milk; ascitic fluid; a paraffin-embedded tissue; gastric juice; gastric lavage fluid;
urine; peritoneal fluid; amniotic fluid; and a cell culture. The method for sampling
the sample, the method for preparing the sample containing a nucleic acid, and the
like are not limited, and, conventional methods known in the art may be employed therefor.
A nucleic acid obtained from such a biological source may be directly used as the
template, or may be used after the sample has been subjected to pretreatment that
modifies the properties of the sample.
For example, in the case in which whole blood is used as the sample, the isolation
of genomic DNA from the whole blood may be carried out by a conventional method known
in the art. For example, a commercially available genomic DNA isolation kit (trade
name: GFX GENOMIC BLOOD DNA PURIFICATION KIT, available from GE Healthcare Biosciences),
etc. may be used.
[0110] Next, a fluorescently-labeled polymorphism detection probe including the fluorescently-labeled
oligonucleotide is added to the sample containing the isolated genomic DNA.
The ftuorescently-labeled probe may be added to a liquid sample containing the isolated
genomic DNA, or may be mixed with the genomic DNA in an appropriate solvent. The solvent
is not particularly limited, and examples of the solvent include conventional solvents
known in the art, such as: a buffer solution such as Tris-HCl; a solvent containing
at least one of KCl, MgCl
2, MgSO
4, or glycerol; and a PCR reaction solution.
[0111] The timing of the addition of the fluorescently-labeled probe is not particularly
limited. For example, in a case in which an amplification process such as PCR described
below is carried out, the ftuorescently-labeled probe may be added to the PCR amplification
products after the amplification process is carried out, or may be added before the
amplification process is carried out.
In the case in which the fluorescently-labeled probe is added before an amplification
process such as PCR is carried out, for example, a fluorescent dye or a phosphate
group may have been added to the 3' end of the probe, as described above.
[0112] The method of amplifying a nucleic acid may be, for example, a method in which a
polymerase is employed. Examples thereof include a PCR method, an ICAN method, a LAMP
method, and an NASBA method. In a case in which the amplification is carried out by
a method in which a polymerase is employed, the amplification may be carried out in
the presence of the fluorescently-labeled probe according to the present invention.
Those skilled in the art would be able to easily adjust the reaction conditions of
the amplification and the like in accordance with the fluorescently-labeled probe
and the polymerase to be used. In the case in which the amplification is carried out
in the presence of the fluorescently-labeled probe according to the present invention,
a polymorphism can be detected by analyzing the Tm value of the ftuorescently-labeled
probe only after the amplification of the nucleic acid is carried out, and, therefore,
it is not necessary to separate the amplification product after completion of the
reaction. Thus, contamination by (or of) the amplification product does not occur.
In addition, since the detection can be carried out by the same apparatus as the apparatus
required for the amplification, conveyance of a vessel is unnecessary, and automatization
of the process is facilitated.
[0113] The DNA polymerase to be used in the PCR method may be selected, without particular
limitation, from DNA polymerases that are usually used for PCR. Examples of the DNA
polymerase include GENE TAQ (trade name, manufactured by NIPPON GENE CO., LTD.), PRIMESTAR
MAX DNA POLYMERASE (trade name, manufactured by Takara Bio Inc.), and a Taq polymerase.
The amount of the polymerase to be used is not particularly limited as long as a usually-applied
polymerase concentration is provided. For example, in the case in which a Taq polymerase
is used, the concentration of the Taq polymerase may be, for example, a concentration
from 0.01 U to 100 U relative to 50 µl of the reaction solution. In this range, for
example, the sensitivity of the detection of the polymorphism in the PIK3CA gene tends
to be increased.
[0114] The PCR method may be carried out under the conditions appropriately selected from
usually-employed conditions.
When the amplification is carried out, the amplification may be monitored using real-time
PCR so that the copy number of the DNA (a sequence to be detected) contained in the
sample can be measured. In other words, the proportion of probes forming hybrids is
increased as the amplification of the DNA (a sequence to be detected) by PCR proceeds,
thereby changing the fluorescence intensity. By monitoring the change in fluorescence
intensity, the copy number and/or the abundance ratio of the sequence to be detected
(either a normal DNA or a mutant DNA) contained in the sample can be obtained.
[0115] In the polymorphism detection method according to the present invention, the fluorescently-labeled
oligonucleotide and a single-stranded nucleic acid in the sample are brought into
contact with each other, thereby allowing hybridization to occur. The single-stranded
nucleic acid in the sample can be prepared by, for example, dissociating the PCR amplification
products obtained in the above-described manner.
[0116] The heating temperature employed for dissociation of the PCR amplification products
(the heating temperature in the dissociation process) is not particularly limited
as long as it is a temperature at which the amplification products can be dissociated.
For example, the heating temperature may be in the range from 85°C to 95°C. The heating
time is not particularly limited, either. The heating time may be, for example, in
the range from 1 second to 10 minutes, or from 1 second to 5 minutes.
[0117] The hybridization of the dissociated single-stranded DNA and the fluorescently-labeled
oligonucleotide may be carried out by, for example, decreasing, after the dissociation
process, the temperature from the heating temperature employed in the dissociation
process. The temperature condition for the hybridization may be, for example, in the
range from 40°C to 50°C.
[0118] The volume and concentration of each component in the reaction solution in the hybridization
process are not particularly limited. In regard to specific examples thereof, the
concentration of DNA in the reaction solution may be, for example, a concentration
from 0.01 µM to 1 µM, or a concentration from 0.1 µM to 0.5 µM. The concentration
of the fluorescently-labeled oligonucleotide may be, for example, in a range in which
the above-described addition ratio relative to the DNA is satisfied, and may be, for
example, a concentration from 0.001 µM to 10 µM, or a concentration from 0.001 µM
to 1 µM.
[0119] The resultant hybrid of the single-stranded DNA and the fluorescently-labeled oligonucleotide
is gradually heated, and the change in fluorescence signal caused by the temperature
increase is measured. For example, in the case of using Q PROBE
®, the fluorescence intensity when the probe is hybridized with the single-stranded
DNA is decreased (or quenched) as compared to the fluorescence intensity in the dissociated
state. Therefore, for example, the hybrid emitting decreased fluorescence or the quenched
hybrid may be gradually heated, and the increase in fluorescence intensity caused
by the temperature increase may be measured.
[0120] The temperature range in which the change in fluorescence intensity is measured is
not particularly limited, and the initial temperature may be, for example, a temperature
from room temperature to 85°C, or a temperature from 25°C to 70°C. The final temperature
may be, for example, a temperature from 40°C to 105°C. The rate of temperature increase
is not particularly limited, either, and may be, for example, in the range from 0.1°C/sec
to 20°C/sec, or in the range from 0.3°C/sec to 5°C/sec.
[0121] Next, the change in signal is analyzed to determine the Tm value. More specifically,
the Tm value may be determined by calculating a differential value at each temperature
(-d(Fluorescence Intensity)/dt) from the fluorescence intensity obtained, and taking
the temperature at which the differential value has the lowest value as the Tm value.
The Tm value may alternatively be determined as the point at which the increase in
fluorescence intensity per unit time ((Increase in Fluorescence Intensity)/t) has
the largest value. On the contrary, in the case in which a probe in relation to which
the signal intensity is increased by the formation of the hybrid, rather than a quenching
probe, is used as the fluorescently-labeled probe, the signal analysis and the determination
of the Tm value may be carried out by measuring the decrease in fluorescence intensity.
[0122] In the present invention, the change in fluorescence signal caused by the temperature
increase (preferably an increase in fluorescence intensity) may be measured while
heating the hybrid as described above. However, instead of this method, the measurement
of the change in signal may alternatively be carried out, for example, during the
course of hybrid formation. In other words, the temperature of the sample, to which
the probe has been added, may be decreased, and the change in fluorescence signal
caused by the temperature decrease may be measured during the course of hybrid formation.
[0123] For example, in the case in which Q PROBE
® is used, the fluorescence intensity is high when the probe is added to the sample
since the probe is in the dissociated state. However, when the hybrid is formed by
a decrease in temperature, the fluorescence is decreased (or quenched). Therefore,
for example, a decrease in fluorescence intensity caused by the temperature decrease
may be measured while gradually decreasing the temperature of the heated sample.
On the other hand, in the case in which a probe in relation to which the signal therefrom
is increased by hybrid formation is used, the fluorescence intensity is low (or quenched)
when the probe is added to the sample since the probe is in the dissociated state.
However, when the hybrid is formed by a decrease in temperature, the fluorescence
intensity is increased. Therefore, for example, the increase in fluorescence intensity
caused by the temperature decrease may be measured while gradually decreasing the
temperature of the sample.
[0124] <Method of Evaluating Drug Efficacy>
The method of evaluating the efficacy of a drug according to the present invention
includes detecting a polymorphism in the PIK3CA gene by the above-described polymorphism
detection method; and evaluating the tolerance to an anticancer agent or the efficacy
of an anticancer agent based on the results of the detection.
In the above-described method of detecting a polymorphism, a polymorphism in the PIK3CA
gene is detected easily with high sensitivity by using the polymorphism detection
probe according to the present invention; therefore, based on this polymorphism in
the PIK3CA gene, an anticancer agent can be evaluated easily with high sensitivity.
[0125] In addition, the tolerance to an anticancer agent and its efficacy can be evaluated
based on the presence or absence of a polymorphism and the abundance ratio of a mutant
sequence and/or a normal sequence. Furthermore, the method of evaluating the efficacy
of an anticancer agent according to the present invention is useful in determining
whether a therapeutic strategy should be changed by, for example, increasing the dosage
of the anticancer agent or using a different anticancer agent, based on the presence
or absence of a mutation and the abundance ratio of a mutant sequence.
Here, specific examples of the anticancer agent to be evaluated include cetuximab
and panitumumab.
[0126] <Reagent Kit for Detecting Polymorphism>
The reagent kit for detecting a polymorphism in the PIK3CA gene according to the present
invention includes the above-described polymorphism detection probe(s).
This reagent kit for detecting a polymorphism includes at least one of the above-described
polymorphism detection probes capable of easily detecting, in the PIK3CA gene, at
least one polymorphism selected from the group consisting of G1624A mutation, G1633A
mutation and A3140G mutation with high sensitivity; therefore, for example, the reagent
kit is able to detect a polymorphism in the PIK3CA gene more easily.
[0127] In addition, the reagent kit for detecting a polymorphism according to the present
invention may also include a primer for amplifying a base sequence containing the
above-described PIK3CA gene polymorphism(s) to be detected. This enables the reagent
kit for detecting a polymorphism according to the present invention to detect a polymorphism
in the PIK3CA gene with good accuracy.
Here, with regard to the probe and primer that may be included in the reagent kit
for detecting a polymorphism, those matters described above can be applied as they
are.
[0128] In a case in which two or more types of fluorescently-labeled oligonucleotide are
contained as the probes, the oligonucleotides may be contained as a mixture, or may
be contained separately.
The two or more types of ftuorescently-labeled oligonucleotide may be respectively
labeled with fluorescent dyes having different emission wavelengths from each other.
By using probes labeled with respectively different fluorescent dyes, detection of
the signal from each ftuorescently-labeled oligonucleotide can simultaneously be carried
out even in a single reaction system.
[0129] Besides the probe and the primers, the reagent kit according to the present invention
may further include reagents required for carrying out nucleic acid amplification
in the detection method according to the present invention. The probe, the primers
and other reagents may be separately contained, or some of them may be contained as
a mixture.
The term "separately contained" may refer to a state in which individual reagents
are separated from each other such that the non-contact state therebetween is maintained,
and does not necessarily require that the individual reagents be contained in separate
containers that can be independently handled.
When the reagent kit includes a primer set for amplifying a base sequence including
a base at the polymorphism site (a region to which the probe can hybridize), detection
of the polymorphism with higher sensitivity, for example, can be achieved.
[0130] The reagent kit according to the present invention may further include an instruction
manual that describes instructions for the generation of a differential melting curve
for a sample containing a nucleic acid to be detected using the PIK3CA probe, and
for the detection of a polymorphism in a gene-encoding base sequence through Tm value
analysis based on the differential melting curve, or instructions that describes various
reagents that are contained, or may additionally be contained, in the reagent kit.
EXAMPLES
[0131] The present invention will now be described in detail by way of examples. However,
the present invention is not limited to these examples in any way.
[0132] [Example 1]
Using a fully-automated SNP analyzer (trade name: I-DENSY (trademark); manufactured
by ARKRAY, Inc.) and a test reagent having the formulation shown in Table 5 below,
Tm analysis was performed to verify the performance of the respective polymorphism
detection probes having the fluorescently labeled oligonucleotide shown in Table 6.
Here, the type of fluorescent dye is indicated in parentheses for the respective probes.
Further, in Table 6, capital letters indicate the bases representing the polymorphism.
The same applies hereinafter.
As a template, the templates (oligo DNAs) shown in Table 7 were employed.
[0133]
[Table 5]
In 50 µL of reaction solution for Tm analysis: |
|
1 × Gene Taq Universal Buffer (manufactured by NIPPON GENE CO., LTD.) |
|
10 µM probe |
0.2 µM |
5 µM oligo DNA |
0.2 µM |
[0134]
[Table 6]
Forward probe |
Name |
Sequence (5'→3') |
mer |
Tm (Wt) |
Tm (Mt) |
|
SEQ ID NO: |
5T-PIK3CA3140-Wt-F3 |
(TAMRA)-caaatgaatgatgcacAtca-P |
20 |
62 |
56 |
6.0 |
8 |
3T-PIK3CA3140-Wt-F8 |
atgcacAtcatggtggc-(TAMRA) |
17 |
66 |
56.5 |
9.5 |
9 |
T-PIK3CA-3140-F10 |
atgcacAtcatggtgg(c-(TAMRA))t-P |
18 |
65 |
56 |
9.0 |
10 |
Reverse probe |
Name |
Sequence (5'→3') |
mer |
Tm (Wt) |
Tm (Mt) |
|
SEQ ID NO: |
5T-PIK3CA3140-Mt-R9 |
(TAMRA)-cagccaccatgaCgtgc-P |
17 |
57 |
70 |
13.0 |
11 |
5T-PIK3CA3140-Mt-R10 |
(TAMRA)-ccaccatgaCgtgcatca-P |
18 |
55 |
67 |
12.0 |
12 |
5T-PIK3CA3140-Mt-R11 |
(TAMRA)-caccatgaCgtgcatca-P |
17 |
52 |
64 |
12.0 |
13 |
5T-PIK3CA A3140G R1-17 |
(TAMRA)-ccatgaCgtgcatcatt-P |
17 |
50 |
63 |
13.0 |
14 |
5T-PIK3CA3140-Mt-R12 |
(TAMRA)-catgaCgtgcatcattcat-P |
19 |
54 |
64 |
10.0 |
16 |
3T-PIK3CA3140-Mt-R16 |
accatgaCgtgcatcattc-(TAMRA) |
19 |
56 |
66 |
10.0 |
17 |
3T-PIK3CA3140-Mt-R17 |
ccaccatgaCgtgcatc-(TAMRA) |
17 |
54 |
66 |
12.0 |
18 |
3T-PIK3CA3140-Mt-R18 |
cagccaccatgaCgtgc-(TAMRA) |
17 |
58 |
70 |
12.0 |
19 |
35T-PIK3CA-3140-R19 |
(TAMRA)-ccatgaCgtgcatcattc-(TAMRA) |
18 |
55 |
66.5 |
11.5 |
20 |
35T-PIK3CA-3140-R20 |
(TAMRA)-ccaccatgaCgtgcatc-(TAMRA) |
17 |
55 |
68 |
13.0 |
21 |
T-PIK3CA-3140-R21 |
c(c-(TAMRA))atgaCgtgcatcattc-P |
18 |
52 |
64.5 |
12.5 |
22 |
Tm values are actual measurements. |
[0135]
[Table 7]
Oligo DNA for forward probe (template) |
Name |
Sequence (5'→3') |
SEQ ID NO: |
PIK3CA A3140G Wt R (wild-type) |
tccatttttgttgtccagccaccatgaTgtgcatcattcatttgtttcatgaaatactcc |
35 |
PIK3CA A3140G Mt R (mutant-type) |
tccatttttgttgtccagccaccatgaCgtgcatcattcatttgtttcatgaaatactcc |
36 |
Oligo DNA for reverse probe (template) |
Name |
Sequence (5'→3') |
SEQ ID NO: |
PIK3CA A3140G Wt F (wild-type) |
ggagtatttcatgaaacaaatgaatgatgcacAtcatggtggacaacaaaaatgga |
37 |
PIK3CA A3140G Mt F (mutant-type) |
ggagtatttcatgaaacaaatgaatgatgcacGtcatggtggacaacaaaaatgga |
38 |
[0136] The Tm analysis was performed by treating the reaction solution at 95°C for 1 second
and then at 40°C for 60 seconds and subsequently measuring the change in the fluorescence
intensity over time during a period in which the temperature of the solution was increased
from 40°C to 80°C at a rate of 1°C/3 seconds. Since TAMRA was employed as a fluorescent
dye, the excitation wavelength was in the range of 520 nm to 555 nm, and the detection
wavelength was in the range of 585 nm to 700 nm. Based on these wavelengths, the changes
in fluorescence intensity originating from the respective fluorescently labeled probes
were measured.
[0137] The Tm analysis yielded Fig. 2 showing the changes in the fluorescence value of the
respective probes. In Fig. 2, the ordinate indicates the change in the fluorescence
intensity per unit time (increase in the d-fluorescence intensity/t), and the abscissa
indicates the temperature (°C). In Fig. 2, the patterns indicated with diamonds represent
the results obtained by using, as a template, the artificial oligo sequence indicated
in SEQ ID NO:35 having the same base sequence as indicated in SEQ ID NO:4 except that
the 155th base (Y) is T and the results obtained by using the artificial oligo sequence
indicated in SEQ ID NO:37 wherein the 155th base (Y) is A, which is complementary
to T. Also, in Fig. 2, the patterns indicated with squares represent the results obtained
using, as a template, the artificial oligo sequence indicated in SEQ ID NO:36 wherein
the 155th base (Y) is C and the results obtained using the artificial oligo sequence
indicated in SEQ ID NO:38 wherein the 155th base (Y) is G, which is complementary
to C.
[0138] Further, in Fig. 2, (A) shows the results obtained using the probe according to the
present invention indicated in SEQ ID NO:8; (B) shows the results obtained using the
probe according to the present invention indicated in SEQ ID NO:9; (C) shows the results
obtained using the probe according to the present invention indicated in SEQ ID NO:10;
(D) shows the results obtained using the probe according to the present invention
indicated in SEQ ID NO:14; (E) shows the results obtained using the probe according
to the present invention indicated in SEQ ID NO:11; (F) shows the results obtained
using the probe according to the present invention indicated in SEQ ID NO:12; (G)
shows the results obtained using the probe according to the present invention indicated
in SEQ ID NO:13; (H) shows the results obtained using the probe according to the present
invention indicated in SEQ ID NO:16; (I) shows the results obtained using the probe
according to the present invention indicated in SEQ ID NO:17; (J) shows the results
obtained using the probe according to the present invention indicated in SEQ ID NO:18;
(K) shows the results obtained using the probe according to the present invention
indicated in SEQ ID NO:19; (L) shows the results obtained using the probe according
to the present invention indicated in SEQ ID NO:20; (M) shows the results obtained
using the probe according to the present invention indicated in SEQ ID NO:21; and
(N) shows the results obtained using the probe according to the present invention
indicated in SEQ ID NO:22.
[0139] From the results shown in Fig. 2, it was proven that, by using the fluorescently
labeled oligonucleotide according to the present invention as a probe, a polymorphism
at the 155th base of the base sequence indicated in SEQ ID NO:4 or 5 can be detected.
[0140] [Example 2]
Using the nucleic acid mixture shown in Table 8 (4 µl/reaction solution) or purified
human genomic DNA (100 copies/reaction solution) as a sample and an automated SNP
analyzer (trade name: I-DENSY (trademark); manufactured by ARKRAY, Inc.), PCR and
Tm analysis were performed. The formulation of the PCR solution is shown in Table
9.
[0141]
[Table 8]
Sample: |
Number of copies of each plasmid in 4 µL of nucleic acid mixture: |
Plasmid name |
Wt sample |
Mt sample (1) |
Mt sample (2) |
exon9 Wt DNA |
1000 |
14000 |
18940 |
G1624A mt DNA |
0 |
1000 |
60 |
G1633AmtDNA |
0 |
5000 |
1000 |
exon20 Wt DNA |
1000 |
9700 |
9970 |
A3140G mt DNA |
0 |
300 |
30 |
[0142]
[Table 9]
PCR solution |
|
In 50 µL of PCR solution: |
|
1 × PCR buffer |
|
Taq polymerase (in-house product) |
1.88 U |
MgCl2 |
1.5 mmol/L |
dNTP |
0.2 mmol/L |
PIK3CA ex9 F |
0.5 µmol/L |
PIK3CA ex9 R |
1 µmol/L |
PIK3CA A3140G F |
2 µmol/L |
PIK3CA A3140G R |
1 µmol/L |
5T-PIK3CA G1633A F1-19 |
0.1 µmol/L |
5FL-PIK3CA-G1624A-F1 |
0.1 µmol/L |
5PB-PIC3CA A3140G R1 |
0.1 µmol/L |
[0143] The PCR was performed by treating the reaction solution at 95°C for 60 seconds and
then repeating 50 cycles of 95°C for 1 second and 58°C for 30 seconds.
Tm analysis was performed after the PCR by treating the reaction solution at 95°C
for 1 second and then at 40°C for 60 seconds and subsequently measuring the change
in fluorescence intensity over time during a period in which the temperature of the
solution was increased from 40°C to 75°C at a rate of 1°C/3 seconds.
[0144] The polymorphism detection probes and primers are shown in Table 10.
[0145]
[Table 10]
Name |
Sequence (5'→3') |
mer |
SEQ ID NO: |
Primer |
|
|
|
PIK3CA ex9 F |
gaacagctcaaagcaatttctacacgag |
28 |
23 |
PIK3CA ex9 R |
cagagaatYtccattttagcacttacYtgtgac |
33 |
24 |
PIK3CA A3140G F |
gaggctttggagtatttcatgaaacaaatg |
30 |
25 |
PIK3CA A3140G R |
gcatgctgtttaattgtgtggaagatccaatc |
32 |
26 |
Probe |
|
|
|
5T-PIK3CA G1633A F1-19 |
(TAMRA)-ctctctGaaatcactAagc-P |
19 |
6 |
5FL-PIK3CA-G1624A-F1 |
(BODIPY FL)-ctctctAaaatcactGagc-P |
19 |
7 |
5PB-PIC3CA A3140G R1 |
(Pacific Blue)-ccatgaCgtgcatcatt-P |
17 |
14 |
[0146] The fluorescent dye, PACIFIC BLUE, has an excitation wavelength in the range of 365
nm to 415 nm and a detection wavelength in the range of 445 nm to 480 nm. The fluorescent
dye, BODIPY FL, has an excitation wavelength in the range of 420 nm to 485 nm and
a detection wavelength in the range of 520 nm to 555 nm. The fluorescent dye, TAMRA,
has an excitation wavelength in the range of 520 nm to 555 nm and a detection wavelength
in the range of 585 nm to 700 nm. Based on these wavelengths, the changes in fluorescence
intensity originating from the respective fluorescently labeled probes were measured.
[0147] The Tm analysis yielded Fig. 3 showing the changes in the fluorescence value of the
respective probes.
In Fig. 3, the ordinate indicates the change in fluorescence intensity per unit time
(increase in d-fluorescence intensity/t), and the abscissa indicates the temperature
(°C).
[0148] With regard to the A3140G mutation, one peak was observed at about 45°C for the wild-type
sample (Wt sample); however, in the samples containing mutant-type plasmids (Mt sample
(1) and Mt sample (2)), another peak was also observed at 57°C. With regard to the
G1624A mutation and the G1633A mutation, one peak was observed at about 48°C for the
wild-type sample (Wt sample); however, in the samples containing mutant-type plasmids
(Mt sample (1) and Mt sample (2)), another peak was also observed at about 55°C.
From the above-described results, it was demonstrated that a polymorphism in the PIK3CA
gene can be detected by using the probe according to the present invention.
[0149] [Comparative Example 1]
Using a fully-automated SNP analyzer (trade name: I-DENSY (trademark); manufactured
by ARKRAY, Inc.) and a test reagent having the formulation shown in Table 11 below,
Tm analysis was performed to verify the performance of the respective polymorphism
detection probes having the fluorescently labeled oligonucleotide shown in Table 12.
Here, the type of fluorescent dye is indicated in parentheses for the respective probes.
As a template, the templates (oligo DNAs) shown in Table 13 were employed.
[0150]
[Table 11]
In 50 µL of reaction solution for Tm analysis: |
|
1 × Gene Taq Universal Buffer (manufactured by NIPPON GENE CO., LTD.) |
|
10 µM probe |
0.2 µM |
5 µM oligo DNA |
0.2 µM |
[0151]
[Table 12]
Forward probe |
Name |
Sequence (5'→3') |
mer |
Tm (Wt) |
Tm (Mt) |
|
SEQ ID NO: |
5T-PIK3CA A3140G F1-16 |
(TAMRA)-cacGtcatggtggctg-P |
16 |
64 |
67 |
3 |
27 |
5T-PIK3CA3140-Wt-F5 |
(TAMRA)-cAtcatggtggctggac-P |
17 |
65 |
61 |
4.0 |
28 |
3T-PIK3CA3140-Wt-F6 |
cAtcatggtggctggacaac-(TAMRA) |
20 |
70 |
68 |
2.0 |
29 |
3T-PIK3CA3140-Wt-F7 |
acAtcatggtggctggac-(TAMRA) |
18 |
68 |
64 |
4.0 |
30 |
3T-PIK3CA3140-Wt-F9 |
caaatgaatgatgcacAtc-(TAMRA) |
19 |
61 |
59 |
2.0 |
31 |
Reverse probe |
Name |
Sequence (5'→3') |
mer |
Tm (Wt) |
Tm (Mt) |
|
SEQ ID NO: |
5T-PIK3CA3140-Mt-R7 |
(TAMRA)-catttttgttgtccagccaccatgaCg-P |
27 |
69 |
73 |
4.0 |
32 |
5T-PIK3CA3140-Mt-R8 |
(TAMRA)-ccagccaccatgaCgtg-P |
17 |
56 |
68.5 |
12.5 |
33 |
3T-PIK3CA3140-Mt-R15 |
aCgtgcatcattcatttgtttc-(TAMRA) |
22 |
64 |
65 |
1.0 |
34 |
Tm values are actual measurements. |
[0152]
[Table 13]
Oligo DNA for forward probe (template) |
Name |
Sequence (5'→3') |
SEQ ID NO: |
PIK3CA A3140G Wt R (wild-type) |
tccatttttgttgtccagccaccatgaTgtgcatcattcatttgtttcatgaaatactcc |
35 |
PIK3CA A3140G Mt R (mutant-type) |
tccatttttgttgtccagccaccatgaCgtgcatcattcatttgtttcatgaaatactcc |
36 |
Oligo DNA for reverse probe (template) |
Name |
Sequence (5'→3') |
SEQ ID NO: |
PIK3CA A3140G Wt F (wild-type) |
ggagtatttcatgaaacaaatgaatgatgcacAtcatggtggctggacaacaaaaatgga |
37 |
PIK3CA A3140G Mt F (mutant-type) |
ggagtatttcatgaaacaaatgaatgatgcacGtcatggtggctggacaacaaaaatgga |
38 |
[0153] The Tm analysis was performed by treating the reaction solution at 95°C for 1 second
and then at 40°C for 60 seconds and subsequently measuring the change in fluorescence
intensity over time during a period in which the temperature of the solution was increased
from 40°C to 80°C at a rate of 1°C/3 seconds. Since TAMRA was employed as the fluorescent
dye, the excitation wavelength was in the range of 520 nm to 555 nm, and the detection
wavelength was in the range of 585 nm to 700 nm. Based on these wavelengths, the changes
in fluorescence intensity originating from the respective fluorescently labeled probes
were measured.
[0154] The Tm analysis yielded Fig. 4 showing the changes in the fluorescence value of the
respective probes. In Fig. 4, the ordinate indicates the change in fluorescence intensity
per unit time (increase in d-fluorescence intensity/t), and the abscissa indicates
the temperature (°C).
In Fig. 4, the patterns indicated with diamonds represent the results obtained using,
as a template, the artificial oligo sequence indicated in SEQ ID NO:35 having the
same base sequence as indicated in SEQ ID NO:4 except that the 155th base (Y) is T
and the results obtained using the artificial oligo sequence indicated in SEQ ID NO:37
wherein the 155th base (Y) is A, which is complementary to T.
Also, in Fig. 4, the patterns indicated with squares represent the results obtained
using, as a template, the artificial oligo sequence indicated in SEQ ID NO:36 wherein
the 155th base (Y) is C and the results obtained using the artificial oligo sequence
indicated in SEQ ID NO:38 wherein the 155th base (Y) is G, which is complementary
to C.
[0155] Further, in Fig. 4, (A) shows the results obtained using the probe indicated in SEQ
ID NO:27; (B) shows the results obtained using the probe indicated in SEQ ID NO:28;
(C) shows the results obtained using the probe indicated in SEQ ID NO:29; (D) shows
the results obtained using the probe indicated in SEQ ID NO:30; (E) shows the results
obtained using the probe indicated in SEQ ID NO:3 1; (F) shows the results obtained
using the probe indicated in SEQ ID NO:32; (G) shows the results obtained using the
probe indicated in SEQ ID NO:33; and (H) shows the results obtained using the probe
indicated in SEQ ID NO:34.
[0156] From the results shown in Fig. 4, it was revealed that, in cases where the polymorphism
detection probe according to the present invention was not used, a polymorphism in
the PIK3CA gene could not be detected because, for example, at least one of the peaks
was obscured or it was difficult to draw a distinction between the wild-type and the
mutant-type due to the small temperature difference between the peaks. It is noted
here that the fluorescently labeled oligonucleotides used in Comparative Example 1
were presented merely as representative examples of undetectable probes that exist
in large numbers.